tag:blogger.com,1999:blog-70051049061743554592024-02-07T09:48:47.691-08:00Principles of Protein StructureA blog principally for present, past and future students of <u><a href="http://www.bbk.ac.uk/">Birkbeck College</a></u>'s Internet-based <a href="http://www.bbk.ac.uk/study/pg/biology/TPCPRSTR.html"><u>Principles of Protein Structure</u></a> course, linking the course to current research and other goings-on in the world of structural molecular biologyDr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.comBlogger88125tag:blogger.com,1999:blog-7005104906174355459.post-56804089605053003932019-07-04T06:56:00.000-07:002019-07-04T08:55:41.128-07:00Birkbeck Science Week 2019: Synthesising LifeBirkbeck holds a Science Week every academic year. In 2019, <a href="http://www.bbk.ac.uk/annual-events/science-week/science-week-2019">Science Week</a> was held in late June, and it kicked off with the Department of Biological Sciences' contribution: a lecture by <a href="http://www.bbk.ac.uk/biology/our-staff/academic/salvador-tomas">Salvador Tomas</a> intriguingly titled ‘Synthesising Life’. Introducing Salvador, the Executive Dean of the School of Science, Nicholas Keep, explained that he had taken both his degrees at the <a href="https://www.uib.eu/">Universitat de les Illes Balears</a> in his native Balearic Islands, before moving to the gloomier climes of the University of Sheffield for postdoctoral study. He set up his own lab at Birkbeck in 2006, and now holds the position of Senior Lecturer in Chemical Biology in the Institute of Structural and Molecular Biology (<a href="http://www.ismb.lon.ac.uk/">ISMB</a>) here.
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The lecture was every bit as engaging as its title suggests. He started by asking the question <i>what is life?</i>, and illustrated the answer by comparing a ‘cyberdog’ with the common-or-garden variety. At a basic level, both dog and cyberdog can be thought of as a network of transistors (or cells) that respond to input signals in different ways, but while the cyberdog is programmed to carry out whatever (presumably) menial tasks its owner demands, the dog is programmed for survival. This led to a formalised definition of ‘life’, as ‘a self-sustained chemical system capable of undergoing Darwinian evolution’. Furthermore, if you zoom in hundreds of millions of times, the dog’s equivalent of the cyberdog’s fundamentally uninteresting network of transistors is the bewildering complexity of ‘<a href="https://en.wikipedia.org/wiki/Molecular_machine">molecular machines</a>’ inside every living cell. Examples of molecular machines that are studied in the PPS course include <a href="http://pps18.cryst.bbk.ac.uk/course/section10/atpsyn.html">ATP synthase</a> and the <a href="http://pps18.cryst.bbk.ac.uk/course/section8/rnatoprotein.html">ribosome</a>.
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<center><i>A cyberdog: Tekno the Robotic Puppy, credit: Toyloverz</i></center>
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The question of ‘how life came to be’ is perhaps almost as old as humanity itself. At the dawn of the scientific age, a few centuries ago, speculations centred on the idea of ‘spontaneous generation’, suggesting that fish might have arisen from water or mice from hay. The development of pasteurisation in the mid-nineteenth century helped disprove this theory, shortly before Darwin published his theory of evolution. We now understand that all living (and extinct) organisms evolved from a simple organism known as <a href="https://en.wikipedia.org/wiki/Last_universal_common_ancestor">LUCA</a> – short for the <b>L</b>ast <b>U</b>niversal <b>C</b>ommon <b>A</b>ncestor – but this begs the question: where did LUCA come from? To find a short answer to this question, you need to go back to the kind of conditions that scientists believe to have existed on an early Earth: a chemically rich ‘warm puddle’ of liquid in an oxygen-poor environment, much like those found in underwater volcanoes today.
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Biochemically, LUCA would have been a single-celled organism containing a minimum set of biomolecules necessary for life, all coded for by a minimal segment of DNA. For decades, scientists have been trying to recreate the process of ‘abiogenesis’ by providing simple molecules in this type of environment with energy and investigating whether more complex molecules, the ‘building blocks’ for LUCA’s DNA and proteins, might be able to form. So far, it has proved possible to make the basic building blocks of proteins, the amino acids, and even, in some circumstances, to join several amino acids into a short chain, but not to connect hundreds of them to form a complete protein. Nucleic acids, the building blocks of DNA, are proving even more intractable.
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Building blocks become biomolecules through a process in which each two units – amino acids or nucleotides – are joined together with the loss of a water molecule. This process requires energy, but the opposite one, in which the bond between the units is broken, can be spontaneous. Salvador used a set of blocks known simply as A, B, C and D to illustrate how the populations of block sets change over time, as combinations such as ‘AB’ are ‘born’ and ‘die’. If AB, for example, is made ‘sticky’ so it attracts more copies of A and B, it becomes ‘autocatalytic’ (that is, it helps form itself) and the AB population burgeons. At least, it does until A or B is depleted, when an ‘extinction event’ occurs. The system becomes more complex with the addition of an energy supply and further building blocks, and it becomes possible to see how collections of units with specialist functions could evolve. Some types would specialise in storing information (the ancestors of nucleic acids) and others in promoting bond formation (the ancestors of proteins).
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<center><i>Building blocks become biomolecules become molecular machines<br>
Top: ATP synthase; Bottom: Bacterial ribosome. From <a href="http://pdb101.rcsb.org/">PDB-101 Molecule of the Month</a>
</i></center>.
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This would be a resourceful molecular system, capable of building its own building blocks, but it would have one major disadvantage: its survival depends on the proximity of the different types of molecule. If it were in the ‘warm puddle’ of the early Earth, a single rainstorm could blow it away. Keeping the components together requires a third type of biomolecule. Lipids are molecules with a long ‘water-hating’ tail and a short ‘water-loving’ head, and in water they form double layers with the tails pointing towards each other. These lipid bilayers often form spherical vesicles, and any primitive biomolecules trapped inside such a vesicle will stay together come what may.
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<a href="https://en.wikipedia.org/wiki/Vesicle_(biology_and_chemistry)">Vesicles</a> containing both ‘DNA-like’ and ‘protein-like’ molecules can be thought of as ‘protocells’: or, if you like, putative ancestors of the ancestors of LUCA. Salvador explained that his own contribution to the evolving story of synthesising life was in exploring the chemistry inside such protocells. Something like a protocell is almost certain to have existed, and this will have evolved to be better programmed for survival through developing more efficient molecular ways of making use of resources, storing and using energy, and responding to stimuli. Reproducing this process by adding molecular machines and efficient, specialist switches to a blank vesicle or protocell can generate cell-like robots. Initially, these are likely to have a wide variety of useful but still quite mundane functions in, for example, <a href="https://en.wikipedia.org/wiki/Targeted_drug_delivery">targeted drug delivery</a>, but eventually they might do more: ‘life, but not as we know it’, perhaps?
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Salvador ended his talk by asking two questions: can we synthesise life, and if so, should we? Most of his audience agreed with him that the first was ‘not done yet, but seems likely in the near future’. Interestingly, however, a majority thought that it might be too risky to take far.
Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com1tag:blogger.com,1999:blog-7005104906174355459.post-69378281461502433312018-12-11T02:15:00.000-08:002018-12-11T02:15:45.747-08:00Atoms and Empty Space: the Structural Biology of SpecificityThe eminent structural biologist <a href="http://www.sussex.ac.uk/profiles/243849">Laurence Pearl</a>, Professor of Structural Biology in the <a href="http://www.sussex.ac.uk/gdsc/">Genome Damage and Stability Centre</a> at the University of Sussex, has strong links with Birkbeck. He studied for his PhD under Professor (now Sir) <a href="https://en.wikipedia.org/wiki/Tom_Blundell">Tom Blundell</a> at Birkbeck in what was then the Department of Crystallography (and the only full university department so named in the UK). The latest of the many honours and awards that he has received is the<a href="http://biochemistry.org/"> Biochemical Society</a>'s prestigious <a href="http://www.biochemistry.org/Awards/TheNovartisMedalandPrize.aspx">Novartis Medal</a> for 2018. This is an annual award, made "in recognition of contributions to the development of any branch of biochemistry" and the winner is invited to present a lecture either at the Society's London headquarters or at one of its many conferences. Pearl gave his Novartis Medal Lecture, entitled <i>Atoms and Empty Space: the Structural Biology of Specificity</i> at the London office of the Biochemical Society on 13 November 2018.
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This lecture was live-streamed on Facebook. It is linked from the <a href="https://www.facebook.com/biochemicalsociety/">Biochemical Society Facebook page</a>, but you don't need to have a Facebook account to view it; just click on the video link below. (It is about an hour long.)
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src="https://www.facebook.com/plugins/video.php?href=https%3A%2F%2Fwww.faceb
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<b>Video © <a href="http://biochemistry.org/">Biochemical Society</a> 2018</b>
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To summarise the lecture briefly, Pearl began with a well-known but still controversial quote from the Greek philosopher <a href="https://en.wikipedia.org/wiki/Democritus">Democritus</a>: "Nothing exists except atoms and empty space. All else is opinion." He then summarised the beginning of his scientific career as a postgraduate student at Birkbeck. During his PhD he learned computer programming and wrote - or contributed to - some of the first structural bioinformatics programs, as well as studying the structure of the aspartic protease, endothiapepsin (PDB <a href="https://www.rcsb.org/structure/4APE">4APE</a>). This was in the early 1980s, when the world was becoming aware of the scourge of AIDS and the necessity of targeting its viral cause, HIV. Sequencing the tiny HIV genome revealed a protease with similar sequence motifs to aspartic proteases, but less than half as long. It was Pearl, by then a postdoc at the <a href="https://www.icr.ac.uk/">Institute of Cancer Research</a> in London, and Willie Taylor from Birkbeck who predicted, years before the structure was solved, that <a href="https://www.ncbi.nlm.nih.gov/pubmed/3306411">HIV's protease would be active as a dimer</a>. (There is more about this in section 7 of PPS, on Quaternary Structure.) The HIV protease structure - exactly as Pearl and Taylor had predicted it - became an important tool for the discovery of protease inhibitors as drugs against AIDS. The first protease inhibitor to enter the clinic was named <a href="https://en.wikipedia.org/wiki/Saquinavir">saquinavir</a> after the amino acid motif SQNI that led them to predict the enzyme's dimeric structure.
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Not surprisingly, this discovery also proved to be a gateway to Pearl's career as an independent researcher. He set up his lab in 1989 in the biochemistry department at <a href="https://www.ucl.ac.uk/biosciences/departments/smb">UCL</a>, with research projects in three areas: DNA repair, signal transduction, and a group of unrelated proteins that help other proteins to fold and that have collectively been named chaperones. He spent the rest of his lecture describing just a few of the many proteins that his group has studied during the last 30 years: first at UCL, then back at the Institute of Cancer Research and since 2009 at Sussex. These included the enzyme uracil DNA glycosylase, which is involved in the specific repair of cytosine residues in DNA that have been deaminated to form uracil. His co-worker on this project was <a href="http://www.bbk.ac.uk/biology/our-staff/academic/renos-savva">Renos Savva</a>, who is now a senior lecturer at Birkbeck and director of our MSc course in Biobusiness. The structure of this enzyme (<a href="http://www.rcsb.org/structure/1UDI">PDB 1UDI</a>) explains the exquisite specificity of this enzyme. Another, much smaller DNA repair enzyme, mismatch uracil glycosyase, was found to have the same fold. His more recent work, which uses electron microscopy as well as X-ray crystallography, includes the structure and mechanism of glycogen synthase kinase, and the structure and dynamics of further DNA damage and repair systems. Listen to the lecture if you want to find out more!Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com3tag:blogger.com,1999:blog-7005104906174355459.post-78172766223690698912018-10-04T10:06:00.001-07:002018-10-04T10:06:44.175-07:00Welcome to new PPS students - and a few more links...This post is very like those I have written at the beginning of the academic year for the past few years; this is because what I have to say now is also very similar...
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I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have recently started studying Birkbeck's <a href="http://pps18.cryst.bbk.ac.uk/">Principles of Protein Structure</a> (PPS) course, and a welcome back to any who have taken a break in studies and intend to complete the course this year. Welcome too if you are thinking that you might want to study with us in the future, or if you are just interested in learning more about a fascinating and fast-moving area of research in molecular biology.
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I run this occasional blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. I might, example, report on talks given in the <a href="http://www.ismb.lon.ac.uk/">ISMB</a> seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The programme for Autumn 2018 has the intriguing title of '<a href="http://www.ismb.lon.ac.uk/wp-content/uploads/2018/09/Autumn2018_ISMBSeminars-1.pdf">Mischievous Microbes</a>'; its themes of microbiology and infectious disease biology have links to some of the later sections of the course. Other posts may be reports from conferences (such as <a href="https://bsg.crystallography.org.uk/">this one</a> at Imperial College, London in December) or summaries of recently published papers in protein structure and allied areas/
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Some earlier posts were written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in <a href="https://principlesofproteinstructure.blogspot.com/2012/03/">this post</a> written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.
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Do, if you get a chance, look through some blog posts from earlier years to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!
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I also work part-time as a freelance science writer, and sometimes I even have a chance to write about structural biology. You might like to follow a set of blog posts I wrote from the International Union of Crystallography's triennial meeting in Hyderabad, India last summer (posts from <a href="http://blogs.iucr.org/crystallites/2017/08/22/">22 August</a> - 6 September). The first entry, featuring a talk by <a href="http://www.bbk.ac.uk/news/birkbeck-fellow-awarded-ewald-prize">Sir Tom Blundell</a>, a former head of the Department of Crystallography at Birkbeck (now part of the Department of Biological Sciences) is perhaps most relevant to PPS. Sir Tom was involved in solving the structure of HIV protease, target of some of the most successful drugs for AIDS, and he went on to found a drug discovery company, <a href="https://astx.com/">Astex</a>. This year I reported on a meeting much nearer at hand (in Liverpool) and, specifically, on one of the most exciting advances in structural biology by X-ray crystallography for some years: <a href="https://physicstoday.scitation.org/do/10.1063/PT.6.1.20180724a/full/">X-ray free electron lasers</a>.
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Finally, the best of luck to new students for the 2017-18 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our <a href="http://www.bbk.ac.uk/study/2018/postgraduate/programmes/TMSBISCL_C">MSc in Structural Molecular Biology</a>.
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Best wishes,
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Dr Clare Sansom <br>
Senior Associate Lecturer, Biological Sciences, Birkbeck <br>
Tutor, Principles of Protein StructureDr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-28945814542585575692018-07-30T09:44:00.001-07:002018-07-30T09:44:30.783-07:00Rosalind Franklin Lecture 2018: Eva Nogales, electron microscopistSince 2016, Birkbeck has held an annual lecture named in honour of perhaps the most famous woman scientist ever to work there: <a href="https://en.wikipedia.org/wiki/Rosalind_Franklin">Rosalind Franklin</a>, whose extraordinary, meticulous experimental work was a necessary part of solving the structure of DNA. This lecture is part of Birkbeck’s commitment to the <a href="https://www.ecu.ac.uk/equality-charters/athena-swan/">Athena SWAN</a> equality initiative, and is it given by a woman scientist distinguished in one of the disciplines represented there.
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The 2018 lecture differed from its predecessors in forming part of both Birkbeck’s annual <a href="http://www.bbk.ac.uk/biology/news/bbk-local?uid=7c83ac95c8ea410c9738e3b6f86a6ff3">Science Week</a> and the eighth <a href="http://www.ismb.lon.ac.uk/biennial-symposium/">ISMB Symposium</a>. The Institute of Structural Molecular Biology (ISMB) is a centre of excellence, founded in 2003 to promote and integrate multi-disciplinary research in molecular, cell and structural biology in Birkbeck and its much larger neighbour, UCL. It holds a varied programme of events for faculty members, research staff and students; symposia, held in ‘even years’, are intensive conferences, generally held over two days and featuring talks from international research leaders.
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This symposium was held over two afternoons on Monday 18 and Tuesday 19 June, with the Rosalind Franklin lecture as the last one on the first day. In planning the symposium, its organisers chose to highlight one technique among all those available for researchers at the ISMB: <a href="https://www.fei.com/life-sciences/XRD-NMR/">electron microscopy</a>, as used to study the atomic structures of large protein complexes and ‘molecular machines’. The Institute’s director, Gabriel Waksman, highlighted Birkbeck’s acquisition of a new and very powerful electron microscope – a Titan Krios – in his introduction as ‘something to celebrate’. According to the <a href="https://www.facebook.com/BirkbeckSchoolofScience/">School of Science Facebook page</a>, Birkbeck’s Department of Biological Sciences is the smallest UK university department to house such a powerful microscope, and it is only through the ISMB that it is able to punch so far above its weight. And the Rosalind Franklin lecturer, <a href="http://cryoem.berkeley.edu/">Eva Nogales</a> from the University of California in Berkeley, was only one of several distinguished proponents of this technique to present their research during the symposium.
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<center><i>Eva Nogales with Prof. Nicholas Keep, Dean of the Faculty of Science at Birkbeck. Photo © Harish Patel, Birkbeck</i></center>
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Few women have achieved as much in electron microscopy research as Nogales. Following a short introduction by Professor Nicholas Keep, Dean of the Faculty of Science at Birkbeck, she began her Rosalind Franklin lecture with thanks. She paid tribute to two of the distinguished researchers present, <a href="http://www.bbk.ac.uk/biology/our-staff/academic/helen-saibil">Helen Saibil</a> and <a href="https://www.mpimf-heidelberg.mpg.de/emeritus_groups/biophysics/holmes/curriculum_vitae">Ken Holmes</a>, describing Saibil, the Bernal Professor of Structural Biology at Birkbeck, as an ‘inspirational’ pioneering woman in electron microscopy. Holmes, who had given one of the previous talks at the symposium, worked with Rosalind Franklin as a PhD student at Birkbeck in the 1950s and went on to make ground-breaking discoveries about the structure of the muscle protein, actin.
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Nogales’ main theme during her lecture was her lab’s efforts to decipher the structures of several large, multi-protein complexes that are involved in the process of gene expression. The different types of cells in our bodies – with a few odd exceptions, such as cancer cells – all contain exactly the same DNA in the chromosomes in the cell nucleus. What makes a brain cell differ from a bone cell or a heart call is how the information carried by the genes on those chromosomes is expressed in the functional molecules, mainly proteins. Only a fraction of the genes in a genome are expressed in a given cell at any particular time. <a href="https://en.wikipedia.org/wiki/Gene_expression">Gene expression</a> is the term given to this incredibly complex and exquisitely sensitive process, which can be divided into two stages expressed simplistically as ‘DNA to RNA’ and ‘RNA to protein’. Work in the Nogales lab focuses on two protein complexes that are involved in the first sub-process, the transcription of the DNA sequences of genes into RNA. These bear the rather cumbersome names of polycomb repressive complex 2 (<a href="https://en.wikipedia.org/wiki/PRC2">PRC2</a>) and transcription factor II D (<a href="https://en.wikipedia.org/wiki/Transcription_factor_II_D">TFIID</a>).
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If the DNA in each human chromosome could be stretched out it would measure tens of centimetres in length. It is packed and compressed to fit into the microscopic cell nucleus by winding around histone proteins to form circular units of structure called <a href="https://en.wikipedia.org/wiki/Nucleosome">nucleosomes</a>. Proteins in the ‘transcription machinery’ can only access the DNA to start gene expression if these are loosely packed. PRC2, as its full name implies, represses this process: it does so by adding methyl groups to the alkaline lysine residues of the histones, making the nucleosomes pack more tightly together. The protein complex therefore forms an ‘on-off switch’ for gene expression. Disrupting its function can lead to the uncontrolled cell growth and multiplication that is characteristic of cancer cells and it is therefore a useful target for the design of anti-cancer drugs.
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Nogales explained that PRC2 is a very large protein complex and that determining its structure using electron microscopy presented a considerable challenge. The first structures, obtained before the ‘<a href="https://www.nature.com/news/the-revolution-will-not-be-crystallized-a-new-method-sweeps-through-structural-biology-1.18335">resolution revolution</a>’ in this technique, could only show separate protein molecules as ‘blobs’: later, better structures that revealed the positions of individual atoms proved that these were ‘accurate but not very precise’. The complex is now known to exist in several distinct structural states and to be able to add methyl groups (‘active’) in two of them. The main difference between these is in the position of one helix, which is bent against the rest of the molecule in the ‘<a href="http://www.rcsb.org/structure/6C23">compact active</a>’ conformation but straightens away from it in the ‘<a href="https://www.rcsb.org/structure/6C24">extended active</a>’ one. PRC2 binds to two protein co-factors in ways that mimic the binding of the flexible ‘tails’ of the histone proteins in methylated and unmethylated forms respectively.
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She then showed some even more impressive structures to explain how the complex interacts with nucleosomes. One complex binds between a pair of nucleosomes, and as long as the DNA that links the two is the right length, binding the first nucleosome positions the second so that the right amino acids are brought into the right position in the PCR2 active site for methylation to occur efficiently.
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The second complex discussed, TFIID, is active exactly when PRC2 is not, as its presence is necessary to begin the process through which DNA is transcribed into RNA. This begins with the step-by-step assembly of proteins close to the position on the DNA where transcription is due to start, forming a ‘<a href="https://en.wikipedia.org/wiki/Transcription_preinitiation_complex">preinitiation complex</a>’. TFIID is the first component of this complex to assemble, and this ‘nucleates’ the complex by recruiting other transcription factors so RNA synthesis can begin. Nogales described distinctive structures of parts of the preinitiation complex obtained by members of her group, finishing by showing some unpublished work on its structure and dynamics that included this vital component. If this fascinating lecture has inspired the many young electron microscopists in the audience as much as Helen Saibil’s work inspired Nogales, then the future of the discipline will be in good – and often female – hands.
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<i>This has been cross-posted with minimal alterations from Birkbeck's<a href="http://blogs.bbk.ac.uk/events/"> Events Blog</a></i>
Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com1tag:blogger.com,1999:blog-7005104906174355459.post-82807456028766002902018-04-12T07:16:00.001-07:002018-04-12T10:17:53.706-07:00A Visit to Diamond Light SourceLast February, a group of Birkbeck students were given the opportunity to visit the UK's synchrotron light source, <a href="http://www.diamond.ac.uk/Home.html">Diamond</a>, located near Didcot in Oxfordshire. Most of the students on the trip were taking the <a href="http://tsmb17.cryst.bbk.ac.uk/">Techniques in Structural Biology</a> (TSMB) course as part of a Master's degree. The majority were studying TSMB as a module of the face-to-face M.Sc. in <a href="http://www.bbk.ac.uk/study/2018/postgraduate/programmes/TMSABIOS_C/">Analytical Bioscience</a>; one was on the distance-learning Structural Molecular Biology M.Sc and a few were studying <a href="http://www.bbk.ac.uk/study/2018/postgraduate/programmes/TMSBIOBS_C/">Bio-Business</a> (which does not include TSMB). This photo shows most of the group with Professor Nick Keep, director of the Structural Molecular Biology course and Dr Katherine Thompson, director of Analytical Bioscience, with part of Diamond's main building in the background.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjveLh0-yQCUwyrh2jM5XkNhdyXFXrsVFo-0uxQRW8zXm0NobJt1KtmQWmJ9OD2AvyCllyIDSUkG_dY_f8RiuuXiCjej1y-9O3JTTEDSr18CAmG7Fb3eXUKJ6xo8M9V29hVbbI4V0Exhe4/s1600/WP_20180215_16_41_16_Pro.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjveLh0-yQCUwyrh2jM5XkNhdyXFXrsVFo-0uxQRW8zXm0NobJt1KtmQWmJ9OD2AvyCllyIDSUkG_dY_f8RiuuXiCjej1y-9O3JTTEDSr18CAmG7Fb3eXUKJ6xo8M9V29hVbbI4V0Exhe4/s320/WP_20180215_16_41_16_Pro.jpg" width="320" height="181" data-original-width="1600" data-original-height="903" /></a></div>
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<center><b>Birkbeck staff and students in front of the 'doughnut' that houses the Diamond ring system</b></center>
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Dr David Price from Diamond gave the students a short lecture to introduce Diamond and the types of experiment that can be carried out there.
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He explained that Diamond is a <a href="https://en.wikipedia.org/wiki/Synchrotron_radiation">synchrotron radiation</a> source, which means that it accelerates beams of electrons around the rim of a large doughnut-like structure so they reach speeds approaching that of light and give out extremely intense <a href="https://en.wikipedia.org/wiki/Electromagnetic_radiation">electromagnetic radiation</a> in the form of X-rays 100 billion times more intense than the sun's rays. X-rays have wavelengths that range from 10<sup>-8</sup> to 10<sup>-11</sup> m, and those in the middle of this range, with wavelengths close to the length of inter-atomic bonds. These are ideally suited to probe the structure of matter on the molecular level, and they can do so in a lot of different ways.
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Price explained briefly how a synchrotron radiation source works. Electrons are fired from an 'electron gun' into a linear accelerator and then into a small ring known as the booster synchrotron for further acceleration. They then enter the large storage ring, which has a circumference of about half a kilometre; it is this that gives the overall structure its doughnut-like shape. Strictly speaking, however, it is not a smooth ring but a polygon with 48 straight sections. The electron beam is bent by magnetism at each of its vertices, and it is this process that emits the intense X-rays. The X-rays produced at each vertex are emitted through a 'hole in the wall' at that vertex. They are then filtered and focused using complex optical equipment to give them the exact properties that are needed for a particular experiment. Each of these 'beamlines' is essentially a small laboratory with its own optics and analysis equipment and its own staff team. As the synchrotron structure has 48 vertices, there are 48 possible beamline stations, although they are not all operational at the same time. The Diamond website currently lists <a href="http://www.diamond.ac.uk/Beamlines.html">31 operational beamlines</a>, of which seven have been optimised for macromolecular X-ray crystallography. Some of the other techniques available are absorption and <a href="http://www.chromedia.org/chromedia?waxtrapp=mkqjtbEsHiemBpdmBlIEcCArB&subNav=cczbdbEsHiemBpdmBlIEcCArBP">fluorescence spectroscopy</a> and <a href="https://en.wikipedia.org/wiki/Small-angle_X-ray_scattering">small-angle X-ray scattering</a>.
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There are more than 50 synchrotron radiation sources ('<a href="https://lightsources.org/">lightsources</a>') in the world; Diamond is one of the more powerful and versatile of these, but it is the only one in the UK. Most - about 90% - of its users are academics who are required to publish their work in open access journals; over 1000 publications each year cite Diamond Academics apply for time on a specific beamline, and these applications are peer reviewed. About 10% of users come from industry and pay for access.
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Price also highlighted briefly a few recent examples of research carried out at Diamond, focusing mainly but not exclusively on structural biology. These included the structure of a peptide hormone, GLP-1 (PDB <a href="http://www.rcsb.org/structure/5NX2">5NX2</a>), that stimulates the secretion of insulin by beta cells in the pancreas and that could is an important target for drugs to treat type 2 diabetes. The structure of the EV71 virus (PDB <a href="http://www.rcsb.org/structure/4CDQ">4CDQ</a>), which causes the potentially fatal hand, foot and mouth disease, was solved at Diamond in 2012 and this is now being used to design potential drugs for this occasionally fatal disease. Away from structural biology - but not away from medicine - he described structural studies of metal alloys that can help understand why hip replacements can be rejected and inform the design of improved materials.
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After the talk, the Birkbeck party was divided into two smaller groups to tour of the main site. Both groups visited one of the beamlines devoted to macromolecular X-ray crystallography, <a href="http://www.diamond.ac.uk/Beamlines/Mx/I24.html">I24</a>, where the structure of GLP-1 was solved. This beamline includes facilities for determining structures of viruses and membrane proteins, and for working with very small crystals (down to about 1.5 microns in diameter).
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<center><b>Looking down on a small part of the storage ring</b></center>
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<center><b>Birkbeck staff (Prof. Nick Keep, far left and Dr. Clare Sansom, next left) and students in the control cabin of beamline I24, one of those used for macromolecular X-ray crystallography</b></center>
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The trip ended with a short visit to the UK's high-energy neutron and muon source, located on the same campus as Diamond and named <a href="https://www.isis.stfc.ac.uk/Pages/home.aspx">ISIS</a> after Oxford's river. These beams are also used to probe the structure of matter, using techniques such as neutron diffraction. We would like to thank all Diamond and ISIS staff involved for their contributions to a fascinating day.
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<i>PPS students will learn more about X-ray diffraction in the second-year module Techniques in Structural Molecular Biology (TSMB), and much more still if they choose to take the specialist Protein Crystallography course. And students on either course may get a chance to visit Diamond fir themselves, as we hope to run the trip again next year.</i>Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com2tag:blogger.com,1999:blog-7005104906174355459.post-3530437163683732942018-01-29T14:05:00.000-08:002018-01-29T14:05:07.713-08:00The Joy and Pain of Structural BiologyThe <a href="http://www.crystallography.org.uk/">British Crystallographic Association</a> is, as its name implies, the main organisation supporting crystallograpy and crystallographers in the UK. Theirs is a multi-disciplinary science, and the different needs of the eclectic group of people who call themselves crystallographers are met by the Association's special interest groups: among them, the <a href="http://bsg.crystallography.org.uk/">Biological Structures Group</a> for structural biology. The annual meeting of this group, known as the Winter Meeting, takes place in December, often just a few working days before Christmas. The BSG Winter Meeting has featured on this blog on several occasions: in 2016, it was <a href="https://principlesofproteinstructure.blogspot.co.uk/2017/01/seeing-wood-for-trees-in-structural.html">held at Birkbeck</a> and celebrated the work of one of our distinguished emeritus professors, Steve Wood.
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The 2017 meeting, held in the University of Cambridge's famous <a href="https://www.phy.cam.ac.uk/">Cavendish Laboratory</a>, had a rather unusual theme. The organisers asked each of the invited speakers to talk about the ups and downs of their scientific career - the 'joy and pain' of the meeting title - by focusing on one challenging or important piece of work, perhaps described in a single published paper. Not every speaker managed to keep to just one paper, but all the talks gave useful and at times inspiring insights into how structural biology is done.
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First of all, however, <a href="https://www.phy.cam.ac.uk/directory/longairm">Malcolm Longair</a>, head of the Cavendish Laboratory from 1997 to 2005 and perhaps the only astrophysicist to address the Biological Structures Group, gave a short history of the university's Physics department that was based there and its links to structural and molecular biology. That early history was quite extraordinary; many of the most important advances in atomic and nuclear physics, including the discoveries of the electron and the neutron and the first controlled nuclear disintegrations, were made there. A lab photo taken in 1932 includes no fewer than nine Nobel laureates.
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Crystallography, in those early years, was thought of as part of physics; J.D. Bernal and his group joined the lab in 1931, and the younger Bragg became head of the department in 1938. The rest, as Longair said, was history: seeds of the discipline we now know as structural molecular biology were sown in Bragg's time with Perutz and Kendrew's work on globin structure as well as Watson and Crick's on that of DNA. By the time those studies reached their triumphant conclusion, however, the crystallographers were no longer strictly part of the Cavendish. The 'Unit for Research on the Molecular Structure of Biological Systems’, set up by the Medical Research Council, moved out of the main lab in 1957 into a building known as the 'MRC Hut'. This was the first home of the <a href="https://www2.mrc-lmb.cam.ac.uk/about-lmb/history-of-the-lmb/">MRC Laboratory for Molecular Biology</a> (MRC-LMB) at Cambridge with its enduring reputation for excellent structural biology research.
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The next speaker, Cambridge University's <a href="https://www.bioc.cam.ac.uk/people/uto/blundell">Tom Blundell</a>, began by describing his early career in 'the Other Place': Dorothy Hodgkin's lab at Oxford, where he had shared some of the glory of the insulin structure. He had considered talking about insulin at this meeting, but, he explained, "Dorothy had had the pain of trying to solve the structure for 30+ years... I had the joy of a paper in <i>Nature</i>!" The story he told instead was his group's own: solving the structures of proteins involved in DNA repair. This was a long story, taking in 15 years' worth of papers in <i>Nature</i> (2002, 2010) and <i>Science</i> (2017) and culminating in the 'great joy' of discovering inhibitors validated against an important protein target for oncology.
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DNA damage taking the form of simultaneous breaks in both DNA strands (<a href="https://www.nature.com/scitable/topicpage/repairing-double-strand-dna-breaks-14432332">double-strand breaks</a>) are common but can lead to cell death or cancer. Fortunately, they are easily repaired in healthy cells, mainly through the mechanism of non-homologous end joining (NHEJ). Blundell's group have studied the proteins involved in this complex mechanism for many years. It is a three-stage process, in which the component proteins assemble on the DNA molecule either side of the break; the ends are 'pruned' by adding or removing nucleotides to restore the original sequence and finally, the ends are joined through DNA ligation. One of the proteins involved is a kinase, DNA-PKcs, that exists as a single polypeptide chain of 4128 amino acids. Blundell's group published the structure of this huge molecule in 2010 (<a href="https://www.rcsb.org/structure/3KGV">PDB 3KGV</a>) and it is still the longest single-chain protein to have been solved by X-ray crystallography. Blundell explained that the chain folds into a flexible, circular 'cradle' like structure that can support the DNA double helix, with the ligation taking place inside. The mechanism requires proteins to work as 'stages, scaffolds and steps' to hold the complex together for repair, and his group has solved structures of many other components including the Ku70-Ku80 heterodimer that recognises and binds to the break, initiating the repair, and a nuclease named Artemis with 'a nice pocket for drug discovery'.
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Two talks on structural biology as applied to drug discovery followed. The first was by Pamela Williams from <a href="http://astx.com/">Astex Pharmaceuticals</a>, a company founded by Blundell with Harren Jhoti in 1999 that has just registered its first drug - a kinase inhibitor, Kisqali® (<a href="https://en.wikipedia.org/wiki/Ribociclib">ribociclib</a>) - for clinical use in breast cancer. Williams' talk highlighted another protein family that is just as important in pharmacology as the kinases: <a href="https://www.ebi.ac.uk/interpro/potm/2006_10/Page1.htm">cytochromes P450</a>. We have about 50 different P450 subtypes in our livers, and they catalyse reactions that modify drug molecules so they can be more easily removed from our bodies. A handful of these - the subtypes known as 1A9, 2C9, 2C19 and 3D6 - metabolise most prescription drugs. Human (and all eukaryotic) P450s are <a href="https://en.wikipedia.org/wiki/Integral_monotopic_protein">monotopic membrane proteins</a> with flexible active sites, which allow them to bind a wide variety of substrates but which make the structures hard to solve. Williams' involvement with P450 structural biology began with the first mammalian structure, rabbit cytochrome 2C5, and she joined Astex from California to work on the first human structure, the subtype 2C9. This was published in 2003 (<a href="http://www.rcsb.org/structure/1OG2">PDB 1OG2</a>); a large number of other human structures have followed, yielding useful insights into drug metabolism.
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Ben Bax, who studied for his PhD under Tom Blundell at Birkbeck, has just moved to the University of York after eighteen years at the pharma company GlaxoSmithKline (GSK). His talk described work at GSK to determine the structures of <a href="https://en.wikipedia.org/wiki/DNA_gyrase">bacterial DNA gyrases</a>. These are members of a large class of enzymes called topoisomerases that catalyse topological transitions in DNA; the gyrase, which catalyses DNA supercoiling, is the target of the widely used <a href="https://en.wikipedia.org/wiki/Quinolone_antibiotic">quinolone</a> family of antibiotics (e.g. ciprofloxacin). However, quinolone resistance is increasing, mainly through mutations at specific amino acid positions of the target gyrase. GlaxoSmithKline is investing heavily in the development of novel gyrase inhibitors based on oligonucleotides, and Bax' structural biology group has contributed a large number of still unpublished structures of the enzyme with and without inhibitors or DNA bound to this work.
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<a href="https://www.ebi.ac.uk/about/people/janet-thornton">Janet Thornton</a>, emeritus director of the European Bioinformatics Institute, is one of the best known figures in British bioinformatics. Her talk, on what she termed an 'accidental' paper, took the audience back to the basic principles of protein structure. In the late 70s, when she started her career, there were only about fifteen protein structures known but scientists were already examining those structures to determine characteristic patterns. Many of these first structures determined had major inaccuracies, and discovering and correcting these was a major task for early structural biologists. The <a href="https://en.wikipedia.org/wiki/Ramachandran_plot">Ramachandran Plot</a>, now half a century old, was one of the first tools to be developed to gauge the quality of a protein structure, and it is still widely used. Thornton's 'accidental' (and very highly cited) paper described the program <a href="https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/">PROCHECK</a>, which runs this and other checks on a structure to give a comprehensive assessment of its quality. A PROCHECK record for each structure in the PDB is linked from the database <a href="http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=index.html">PDBsum</a>.
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The final talk provided delegates with a rare opportunity to hear a new Nobel Laureate - in this case, the Laboratory of Molecular Biology's own <a href="https://www2.mrc-lmb.cam.ac.uk/group-leaders/h-to-m/richard-henderson/">Richard Henderson</a> - tell the story behind some of his ground-breaking research. Henderson shared the <a href="https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2017/">2017 Chemistry Nobel</a>, for "developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution", with Joachim Frank and Jacques Dubochet. He chose to talk about one structure that he had in some senses made his own: that of <a href="https://en.wikipedia.org/wiki/Bacteriorhodopsin">bacteriorhodopsin</a>, a proton pump found in Archaea that captures light energy as photons and that has many structural and mechanistic similarities with the G-protein coupled receptors, although the exact evolutionary relationship is unclear. Henderson's studies of this important molecule started in the 1970s with structures that were just about detailed enough to show the cylindrical helices. It took him over 15 years'effort with collaborators in Berlin, Berkeley and elsewhere to improve the technology enough to solve the so-called 'phase problem' and obtain an atomic-resolution structure by electron diffraction. The rest, again, is history.
Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-27240046804966857082017-10-27T04:20:00.000-07:002017-10-27T04:20:40.237-07:00Welcome to new PPS students - and blogging crystallographyThis post is very like those I have written at the beginning of the academic year for the past few years, if posted rather later than usual. This is because what I have to say now is also very similar...
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I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have recently started studying Birkbeck's Principles of Protein Structure (PPS) course, and a welcome back to any who have taken a break in studies and intend to complete the course this year.
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I run this blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. I might, example, report on talks given in the <a href="http://www.ismb.lon.ac.uk/index.html">ISMB</a> seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The programme for Autumn 2017 focuses on the <a href="http://www.ismb.lon.ac.uk/Autumn2017_ISMBSeminarProgramme.pdf">molecular biology of cancer</a>; there is some material on this topic in section 5 of this course, 'Towards Tertiary Structure', where we look briefly at the structure and function of kinases. Many of the newer anti-cancer drugs, including <a href="https://en.wikipedia.org/wiki/Imatinib">Glivec</a>, which has transformed the prospects for patients with chronic myeloid leukaemia, target this class of protein. Other posts may be reports from conferences or summaries of recently published papers in protein structure and allied areas; watch out for one at the end of this year featuring a lecture by the UK's newest Nobel laureate, <a href="http://www2.mrc-lmb.cam.ac.uk/richard-henderson-wins-2017-nobel-prize-chemistry/">Richard Henderson</a>.
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Some earlier posts were written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in <a href="http://principlesofproteinstructure.blogspot.co.uk/2012/03/hello-from-recent-graduate-of.html">this post</a> written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.
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Do, if you get a chance, look through some blog posts from earlier years to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!
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And I hope that you will also be interested in some more blogging of mine, in which I explore crystallography - the most popular experimental technique for determining protein structures - more widely. In August I was lucky enough to attend the congress of the <a href="http://www.iucr.org/iucr">International Union of Crystallography</a> in Hyderabad, India, and to write the conference blog. The topics I covered over the 8 days of the meeting ranged from crystallography in history to crystallography in space, but did also include some structural biology. The <a href="http://blogs.iucr.org/crystallites/2017/08/22/">first post</a> in this series covers the opening ceremony, including the award of one of crystallography's highest honours, the <a href="https://www.iucr.org/iucr/ewald-prize">Ewald Prize</a>, to <a href="http://www.bbk.ac.uk/news/birkbeck-fellow-awarded-ewald-prize">Sir Tom Blundell</a>, a former head of the Department of Crystallography at Birkbeck (now part of the Department of Biological Sciences). Sir Tom is perhaps best known for his part in solving the structure of HIV protease, target of some of the most successful drugs for AIDS, and he went on to found a drug discovery company, <a href="https://astx.com/">Astex</a>. A <a href="http://blogs.iucr.org/crystallites/2017/08/27/">later post</a> describes a plenary lecture by John Spence of Arizona State University on imaging proteins in motion.
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Finally, the best of luck for the 2017-18 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our MSc in Structural Molecular Biology.
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Best wishes,
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Dr Clare Sansom <br>
Senior Associate Lecturer, Biological Sciences, Birkbeck and Tutor, Principles of Protein Structure Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com2tag:blogger.com,1999:blog-7005104906174355459.post-25440531416830200362017-09-06T05:53:00.000-07:002017-09-06T05:53:48.522-07:00The 2017 ISMB RetreatThe Institute of Structural and Molecular Biology (<a href="http://www.ismb.lon.ac.uk/">ISMB</a>) at Birkbeck and UCL holds a conference each summer where all members of the Institute's constituent departments come together to discuss their research. In even years, this takes the form of the ISMB Symposium: two intense days of research talks from ISMB core members and invited research leaders. In odd years, the focus turns to the younger members of the Institute with the <a href="http://www.ismb.lon.ac.uk/retreat.html">ISMB Retreat</a>. This, usually held in Cambridge University's <a href="http://www.robinson.cam.ac.uk/">Robinson College</a>, is a two-day residential event featuring plenaries from outside speakers, shorter talks by students and postdocs from the ISMB, an activity designed to get young researchers thinking about science-based careers outside an academic lab, and a poster session. The 2017 retreat, held on June 29 and 30, was the seventh in the series.
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<b>There is a <a href="http://www.ismb.lon.ac.uk/2017_Retreat_write-up.pdf">full report</a> of this retreat (written by me) with photos on the ISMB website. Do check it out! Here are just a few highlights that are relevant to students of structural biology. </b>
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The three keynote speakers were <a href="http://www2.mrc-lmb.cam.ac.uk/group-leaders/n-to-s/lori-passmore/">Lori Passmore</a> (MRC Laboratory of Molecular Biology (LMB), Cambridge; <a href="https://www.imperial.ac.uk/people/a.rutherford">Bill Rutherford</a> (Imperial College, London); and <a href="http://www.ucl.ac.uk/cancer/research/department-oncology/cell-signalling-group">Bart Vanhaesebroeck</a> (UCL Cancer Institute). Structural biology was a feature of all three talks, although most prominently in Passmore's. She was recently appointed as a group leader at the LMB, where her group uses high resolution electron microscopy to study the process of <a href="http://pps16.cryst.bbk.ac.uk/course/section8/dna2rna3.html">poly-adenylation</a> (link to PPS section 8): the addition of the 'poly-A tail' to newly synthesised messenger RNA molecules during the maturation process. Much of the work she discussed has still to be published, apart from a section on techniques development: a physicist in her group, Chris Russo, has worked with her to devise a novel, gold-based substrate for mounting specimens in the electron microscope that eliminates most specimen movement and thus increases resolution.
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Bill Rutherford gave an engaging talk, unusually featuring hand-written slides, on the mechanism of action of <a href="https://en.wikipedia.org/wiki/Photosystem_II">photosystem II</a>, one of the complex proteins involved in photosynthesis. The evolution of photosynthesis was responsible for the increase in oxygen in the Earth's atmosphere that made multicellular life possible, and Rutherford explained how the increase in oxygen had led to further changes in the structure and mechanism of the photosystem that had the overall effect of decreasing its efficiency. Bart Vanhaesebroeck's lecture, which ended the retreat, described the mechanism of the <a href="https://en.wikipedia.org/wiki/Phosphoinositide_3-kinase">phosphoinositide-3-kinase</a> family of proteins and their role in cancer. <a href="http://pps16.cryst.bbk.ac.uk/course/section5/kinase.html">Inhibitors of these kinases</a> (link to PPS section 5) might prove useful anti-cancer drugs, almost certainly as part of combination therapy.
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The programme also included nine short talks by students and young postdocs. The excellent quality of these was highlighted by the judges of the best talk, who were unable to come to a consensus judgement. In the end, two equal prizes were given, to Jennifer Booker, who studies the structure of sodium channels (see e.g. <a href="http://principlesofproteinstructure.blogspot.co.uk/2016/09/shining-light-on-3d-structures-of.html">this post</a> from September 2016) in <a href="http://people.cryst.bbk.ac.uk/~ubcg25a/">Bonnie Wallace's group</a> at Birkbeck, and to <a href="https://sites.google.com/site/rnaplab/people/sapirofer">Sapir Ofer</a>, for a talk on her PhD studies of the structural and molecular biology of archaeal <a href="https://en.wikipedia.org/wiki/Histone">histones</a>. Proteins in this family are responsible for packing DNA into the chromosomes of eukaryotic cells; bacteria pack DNA using an entirely different mechanism, but the mode of packing in archaea - single-celled organisms that have no nucleus and are defined as prokaryotes but are more closely related to eukaryotes than to bacteria - was unknown until recently.
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The retreat always includes an activity to challenge ISMB students and postdocs to think about career opportunities beyond academic research, and this year's proved particularly popular. It was a <a href="http://www.bbc.co.uk/programmes/b006vq92">Dragons' Den</a> style competition in which the younger delegates were split into teams, assigned mentors and given an hour to create a fictitious life science company, develop a bid for funding and present this to a panel of 'dragons'. Three finalists were chosen to battle it out in front of the rest of the delegates. The competitors worked hard and all 'companies' produced ideas that held water to at least some extent, but there could be only one winner. That was a company named TerraNova, which presented an antibody-drug conjugate for primary progressive multiple sclerosis.
Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-17484000454751651202017-07-18T04:22:00.001-07:002017-07-18T04:24:38.145-07:00Highlights from the summer 2017 ISMB seminar programme
Regular readers of this blog will know that the <a href="http://www.ismb.lon.ac.uk/index.html">Institute of Structural and Molecular Biology</a> (ISMB) coordinates the research efforts in the Department of Biological Sciences at Birkbeck and two departments – Chemistry and Structural and Molecular Biology – at neighbouring University College London. Research in the participating departments is coordinated through six core research centres, and many grants, PhD studentships and experimental facilities are held in common.
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Since 2010, too, these departments’ seminar programmes have been consolidated into termly series of <a href="http://www.ismb.lon.ac.uk/seminar.html">ISMB seminars</a>, giving the Institute’s researchers and students the chance to hear world-class scientists present their work. Most of each term’s seminars are centred round a theme, with recent themes including bioinformatics; the molecular basis of infectious disease; and, in the most recent series, ‘beyond signalling’. This post briefly describes two seminars in this series, both from researchers based in London and both closely concerned with disease mechanisms.
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<a href="https://www.imperial.ac.uk/people/p.freemont">Paul Freemont</a> holds a chair in protein crystallography at Imperial College, London. His group has solved the structure of several proteins linked to cancer, including a domain called the ‘RING finger’ that is found in the breast cancer susceptibility protein BRCA1. His talk to the ISMB, however, concerned a research interest that he shares with the Institute’s head, <a href="http://www.ismb.lon.ac.uk/gabriel-waksman/homepage.htm">Gabriel Waksman</a>: the membrane-bound protein complexes through which Gram negative bacteria secrete toxins and other molecules across the double bacterial cell wall and out of the cell. These bacteria have evolved at least six such ‘molecular machines’, with yet another found in mycobacteria such as <i>M. tuberculosis</i>. Waksman’s work in elucidating the structure of the Type IV system is <a href="http://pps16.cryst.bbk.ac.uk/course/section11/secretion-pili.html">covered extensively</a> in section 11 of the PPS course (‘Structures of Membrane Proteins’).
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Freemont’s seminar described the <a href="https://en.wikipedia.org/wiki/Type_VI_secretion_system">Type VI secretion system</a> (T6SS), which was first identified as recently as 2006 in <i>Vibrio cholerae</i> (as its name suggests, the causative agent of cholera). The function of the T6SS is to eject proteins from the interior of the bacterial cell into an adjacent cell, which may be either bacterial or eukaryotic. Freemont’s lab mainly studies these systems in the bacterium <i>Pseudomonas aeruginosa</i>, an opportunistic pathogen that causes infections mainly in people who are already chronically ill, such as cystic fibrosis patients and those with severe burns.
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This secretion system has been described as a ‘molecular syringe’. Its structure resembles that of the tail of a <a href="https://en.wikipedia.org/wiki/Bacteriophage">bacteriophage</a> – a type of virus that affects bacteria – but it is inverted, with the tip of the tail pointing away from the bacterial cell wall and towards its target cells. In some species, the same secretion systems can target both eukaryotic cells and other bacteria. The system consists of a long sheathed tube, built up from many protein subunits, that is large enough to be easily viewed using electron tomography and that is tipped by a spike through which the protein to be delivered is ejected. Energy for cargo delivery is provided by the contraction of the tube, with a single contraction storing the energy equivalent of 1600 molecules of ATP. The whole structure is dynamic; it is assembled only when needed and disassembled after the cargo has been delivered, allowing the cycle to begin again.
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Although the T6SS can sometimes act as a cell-to-cell ‘killing machine’, as in <i>Vibrio cholerae</i>, protein delivery to the target cell will often have rather more subtle effects, with <i><a href="https://microbewiki.kenyon.edu/index.php/Pseudomonas_aeruginosa">Pseudomonas aeruginosa</a></i> a case in point. This rod-shaped Gram negative pathogen uses three distinctly different type 6 systems, encoded on separate operons, to secrete effector proteins that interfere with the host immune system. Freemont and his group have solved the structures of several <i>P. aeruginosa</i> T6SS components using X-ray crystallography, throwing more light on their phage-like mechanism of action. Structures of an accessory protein (<a href="https://www.rcsb.org/pdb/explore.do?structureId=4UQX">TagJ</a>) and the ATPase that catalyses sheath disassembly (<a href="https://www.rcsb.org/pdb/explore.do?structureId=4UQW">ClpV</a>) were all published in the <i><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239648/">Journal of Biological Chemistry</a></i> in 2014; some other component structures are yet to be published. TagJ is now known to interact both with ClpV, an AAA+ ATPase, and with components of the sheath, and this interaction allows the rapid disassembly that is required for the complete system to be reset. Each ATPase only interacts with the components from its own operon. Further structural studies, using high-resolution electron microscopy as well as X-ray crystallography, are expected to elucidate further details of these complex molecular machines and to suggest ways in which they might one day be targeted by the novel antimicrobial drugs that we so desperately need.
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The second London-based seminar speaker, <a href="https://www.westminster.ac.uk/about-us/our-people/directory/dwek-miriam">Miriam Dwek</a> from the University of Westminster, had a somewhat unorthodox beginning to her research career at Oxford University’s first spin-off company, Oxford Glycosystems (now, after many mergers, part of pharma giant <a href="http://www.ucbpharma.co.uk/home">UCB</a>). She has maintained her interests in glycobiology (the biology and biochemistry of sugars and polysaccharides) and its application to human disease – particularly breast cancer – into and throughout her academic research career.
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<a href="http://www.breastcanceruk.org.uk/">Breast cancer</a> is one of the most common cancer types, with 400,000 new cases occurring each year in Europe alone. Breast tumours can be divided into many subtypes with different genetic and biochemical profiles; although some are now easily treated with surgery, radiotherapy and/or drugs, others are often fatal (if perhaps after many years). Generally speaking, tumours are tractable when they are confined to breast tissue and the disease only becomes difficult to treat once it has spread. All cancer types metastasise in a particular pattern; breast cancers tend to spread first into nearby lymph nodes and then to the lungs, brain or bones.
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<a href="https://en.wikipedia.org/wiki/Metastasis">Metastasis</a> is a complex, multi-step process, and selecting the optimum treatment for each patient depends on detecting whether and how her tumour will metastasise as early as possible. Changes in the concentration of some biological molecules in body fluids have been associated with tumour growth and development, and these biomarkers can be used as easily-measurable surrogates of cancer development. One particularly well-known example is the prostate-specific antigen (PSA), a glycoprotein found in semen that is elevated in prostate cancer. No such clear-cut examples exist in breast cancer, but many subtler biochemical changes are known to occur. Dwek and her group have been exploring differences in protein glycosylation patterns between breast tumours and normal breast cells.
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Glycosylation is one of the most common <a href="http://pps16.cryst.bbk.ac.uk/course/section2/ptm.html">post-translational modifications</a> of amino acids (link is to PPS section 2). There are two basic types; one or (almost always) more monosaccharides can be bonded to the oxygen atoms of serine and threonine side chains (O-glycosylation) or to asparagine’s side chain nitrogen (N-glycosylation). The addition of the first residue to the amino acid and the subsequent extension of the chain are catalysed by enzymes in the <a href="http://pps16.cryst.bbk.ac.uk/course/section10/enz_over.html">transferase</a> class. In O-glycosylation, in particular, the patterns of residues added to the glycan ‘branches’ differ between healthy breast epithelial cells and breast tumour cells, and this, in turn, can aid the process of cell adhesion (binding cells together), which is essential for tumour metastasis. <a href="https://en.wikipedia.org/wiki/Cadherin">Cadherins</a> are glycoproteins that have important roles in cell adhesion, and Dwek’s group used <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3013464/">glycoproteomics</a> techniques to identify this as a potential biomarker of metastatic breast cancer. She considers that it is likely to be particularly useful for detecting metastasis in patients with estrogen receptor positive tumours and vascular invasion.
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Other topics presented by leading researchers in this ISMB seminar series included nuclear receptors, collagen-binding proteins and protein targeting and translocation. The seminar programme will return in October, and I will doubtless be returning to it again in this blog.
Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-27090969256980506712017-03-07T06:07:00.000-08:002017-03-07T06:07:57.193-08:00Mapping the Evolution of Enzyme FunctionThe <a href="http://www.ismb.lon.ac.uk/">Institute of Structural and Molecular Biology</a>, which combines the research endeavours of Birkbeck and University College London in these disciplines, runs a programme of weekly research seminars throughout the university terms. Each term’s seminars are linked by a theme, and the theme for the spring term of 2017 has been ‘<a href="http://www.ismb.lon.ac.uk/Spring-2017_ISMBSeminarProgramme.pdf">Bioinformatics and Computational Biology</a>’. Early in February, the Institute was delighted to welcome one of the UK’s foremost structural biologists, <a href="http://www.ebi.ac.uk/about/people/janet-thornton">Professor Dame Janet Thornton</a>, to give a talk in this series. Thornton was well known to many in the large audience, having spent the whole of the 1980s at Birkbeck, rising to be a professor in the School of Crystallography (now part of Biological Sciences). During the 1990s she held chairs at both Birkbeck and UCL and founded a biotech company, Inpharmatica, before leaving to direct the <a href="http://www.ebi.ac.uk">European Bioinformatics Institute</a> (EBI) at Hinxton, near Cambridge. She has now stepped down from the directorship but maintains an active research group at the EBI.
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The topic that Thornton chose to present was one that she had worked on throughout her long career: the structure, function and evolution of the enzymes. When she started studying proteins there were probably about 20 known structures. The <a href="http://www.rcsb.org/pdb/home/home.do">PDB</a> now holds well over 120,000 protein structures, and tens of thousands of these are of enzymes, so there is plenty of data to work with.
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And enzymes are particularly easy to work with because their functions are so well characterised. Back in the 1960s an <a href="https://en.wikipedia.org/wiki/Enzyme_Commission_number">Enzyme Commission</a> assigned a set of four numbers (‘EC numbers’) to each enzyme. There are six primary enzyme classes, each of which is divided into sub-classes and sub-sub-classes; the final number is a serial number that defines the enzyme’s substrate. So, for example, <a href="http://enzyme.expasy.org/EC/3.1.4.11">phosphoinositide phospholipase C</a> is also known as EC 3.1.4.11; the 3 indicates that this enzyme is a hydrolase, the 1 that it acts on ester bonds and the 4 that it is a phosphoric diester hydrolase. The other top-level classes are the oxidoreductases (1); the transferases (2); the lyases (4); the isomerases (5); and the ligases (6). EC numbers define enzyme function rigorously, so referencing them in computer programs is straightforward.
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Thornton and her group chose to focus on those enzymes that have a well-characterised catalytic function that is mainly involved in small-molecule metabolism. All enzymes with these characteristics were grouped into homologous superfamilies (that is, families of proteins with a clear evolutionary ancestor) and the members of each superfamily were annotated with EC numbers as a proxy for their function. For example, the superfamily of enzymes that are clearly related to phosphoinositide phospholipase C by structure and function includes not only enzymes classified as 3.1.4.11 but also sphingomyelin phosphodiesterases D (<a href="http://enzyme.expasy.org/EC/3.1.4.41">3.1.4.41</a>) and phosphatidylinositol diacylglycerol-lyases (<a href="http://enzyme.expasy.org/EC/4.6.1.13">4.6.1.13</a>). The two phosphodiesterases have the same chemistry (as specified by the first three EC numbers) but act on substrates with very different shapes, while the chemistry of the enzymes 4.6.1.13 differs significantly from the others.
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In this example, comparing the structures of enzymes with the EC numbers 3.1.4.41 and 3.1.4.11 showed that active site residues involved in their reaction mechanism and the bound metal ion in each one that is necessary for catalysis superimpose very well, but the rest of the active site varied significantly to allow substrates with distinctly different sizes and shapes to bind. In contrast, the lyase 4.6.1.13 has a similar-shaped active site to 3.1.4.11 but no bound metal and different catalytic residues. In this case is likely that a single amino acid change, removing an aspartic acid residue and therefore a negative charge, has removed the ability of the enzyme to bind a metal ion and thus changed the reaction that the enzyme catalyses.
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Enough data was available to group the enzymes in this superfamily, and in another 275, into phylogenetic trees to map out the evolutionary route taken within each superfamily and catalogue all possible evolutionary changes of function. Some of these are much more complex than the one outlined above. For example, the analysis showed that five classes of flavin-dependent mono-oxygenases with different chemistry were evolutionarily related. Here, the change in chemistry seems to have arisen not from a simple substitution of one amino acid for another but a change in the multi-domain architecture of the protein.
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The group constructed an ‘EC exchange matrix’ from this data to show how many times each top-level EC class had changed into each other class during evolution. While most changes in chemistry left the top-level class – the basic type of the reaction – unchanged, every possible change had occurred at least once in evolutionary history. In fact, 11% of the changes catalogued were changes to top-level class. The diagram below illustrates this data in a series of six circles, one for each ‘original’ enzyme class, with the width of each strip indicating the number of transitions from one class to another: for example, the thick red strip going from the ‘top’ to the ‘bottom’ of the top left-hand circle illustrates that a lot of transitions from oxidoreductases (class 1) to transferases (class 2) have been observed.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEggX1K8ZZqpj2vjIpnPRm15vWxEoLzBCaIjuskvnakbg9CoN5nYH4CU6lHwarm9AOFD1-tBW4rypiZ3fTJzTPW-2Iice9lAaDoSuFGKGAB4tK-0OdSjIIZL56xxjiUnioRBvlLaLnOt-Qo/s1600/functional+evolution.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEggX1K8ZZqpj2vjIpnPRm15vWxEoLzBCaIjuskvnakbg9CoN5nYH4CU6lHwarm9AOFD1-tBW4rypiZ3fTJzTPW-2Iice9lAaDoSuFGKGAB4tK-0OdSjIIZL56xxjiUnioRBvlLaLnOt-Qo/s320/functional+evolution.jpg" width="320" height="169" /></a></div>
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<b>An overview of functional evolution in enzymes. © Nicholas Furnham & Sergio Martinez Cuesta, EBI</b>
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They then looked in much more detail at the changes observed in the catalytic site of each superfamily during evolution, and found that active sites differ in ‘plasticity’. At one extreme there is the <a href="http://pps16.cryst.bbk.ac.uk/course/section5/mainalpha.html">TIM barrel</a> ‘superfold’, which is a scaffold that holds amino acids with different chemistry in similar positions to catalyse many different reaction types. At the other extreme, there are seven superfamilies in which the catalytic residues are 100% conserved. It is interesting to try to correlate sequence similarity with ‘functional similarity’, but this runs into the problem of how to define functional identity. With enzymes, any measure of functional similarity will include a contribution from the chemical similarity of the substrates and this is difficult to gauge, particularly as most of the best computational tools were written for commercial drug discovery and are therefore not in the public domain. Preliminary results suggest that there is some correlation, but it is much weaker than that between sequence and structural similarity.
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Thornton summed up her lecture by re-stating that evolutionary changes to enzyme substrate specificity are much commoner than those to basic chemistry. Evolution has, however, given rise to an explosion in enzyme function. The EC system has catalogued a total of 2,994 unique enzyme functions, but only 379 different structures (CATH superfamilies) are known to have enzymatic activity. Most enzyme functions will therefore have evolved from another function, with each catalytic activity arising independently only a few times throughout evolutionary history. The evolutionary relationships within enzyme superfamilies are complex and there are many ways in which their function can diverge.
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Much of the work Thornton presented has been described in a 2012 paper in <i><a href="http://europepmc.org/articles/PMC3291543">PLoS Computational Biology</a></i>; its lead author, <a href="http://www.lshtm.ac.uk/aboutus/people/furnham.nick">Nick Furnham</a> from the Thornton group at the EBI, is now a group leader at one of Birkbeck’s neighbouring colleges, the <a href="http://www.lshtm.ac.uk">London School of Hygiene and Tropical Medicine</a>. PPS students will learn much more about the structure, function and mechanisms of enzymes in section 10 of the course, ‘Protein Interactions and Function’.
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The most recent paper from the Thornton group on this topic is: <br>
Furnham N, Dawson NL, Rahman SA, Thornton JM, Orengo CA. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751976/">Large-Scale Analysis Exploring Evolution of Catalytic Machineries and Mechanisms in Enzyme Superfamilies</a>. <i>Journal of Molecular Biology</i> <b>428</b> (2016) p.253-267
Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-8284360444613018452017-01-24T07:32:00.001-08:002017-02-07T04:18:19.051-08:00Seeing the Wood for the Trees in Structural BiologyThe <a href="http://www.crystallography.org.uk/">British Crystallographic Association</a> (BCA) was set up in 1982 to support UK scientists working in crystallography and other structure-based sciences. It has five specialist groups (four discipline-based, and one for young crystallographers): the <a href="http://bsg.crystallography.org.uk/">Biological Structures Group</a> for structural biologists holds its main annual conference each December, generally just before the Christmas break. Several of these one-day Winter Meetings have been previously described in this blog. The <a href="http://bsg.crystallography.org.uk/wint16.html">2016 meeting</a>, however, was particularly relevant for anyone connected to Birkbeck: not only was it held in the college, but it celebrated the work of one of the college’s most distinguished structural biologists, Steve Wood. The meeting title was, of course, a pun on his name.
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Wood worked with <a href="http://www.bioc.cam.ac.uk/people/uto/blundell">Professor Sir Tom Blundell</a> at Birkbeck in the 1990s to solve the structure of an important small human protein, serum amyloid P component (SAP or pentraxin; PDB <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=1SAC">1SAC</a>). This protein forms pentamers that bind to amyloid fibres and it is thought to be involved in the protection of those fibres from breakdown by proteases. Pentraxin-binding compounds that interfere with this process might be useful as treatments for amyloidosis and other diseases associated with protein aggregation, perhaps including Alzheimer’s disease.
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Blundell, a former head of Birkbeck’s Crystallography Department and now emeritus professor of Biochemistry at the University of Cambridge, kicked off the meeting in fine style. He had known Wood since they were, respectively, a young lecturer and a PhD student at the University of Sussex in the 1970s, and they have published over 60 papers together. His talk surveyed the structural biology of multi-protein signalling systems over the last 40 years. The earliest such system to be discovered involved the control of blood sugar levels through insulin and glucagon binding to their receptors. The general principles developed through structural studies of this relatively simple system have been applied to other, more complex ones including the interaction between the breast cancer susceptibility protein BRCA2 and a recombinase enzyme that controls one type of DNA repair. Mutations that interfere with this binding lead to greatly enhanced susceptibility to some cancer types. Blundell’s group at Cambridge set up a database, <a href="http://marid.bioc.cam.ac.uk/credo">CREDO</a>, to catalogue the interactions involved in all macromolecular complexes in the PDB. Many protein-protein interactions are now actual or potential drug targets. Some promising drugs for solid tumours act by inhibiting the interactions between cyclins and cyclin dependent kinases (CDKs) that drive cells through the cell cycle. <a href="http://astx.com/">Astex Pharmaceuticals</a>, the drug discovery company set up by Blundell and some of his Cambridge colleagues in 1999, has one such CDK inhibitor – ribociclib – that has completed <a href="https://www.novartis.com/news/media-releases/monaleesa-2-trial-novartis-lee011-ribociclib-stopped-due-positive-efficacy">Phase III clinical trials</a> for advanced breast cancer.
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<a href="http://biology.st-andrews.ac.uk/contact/staffProfile.aspx?sunid=glt2">Garry Taylor</a>, who gave the next talk, joined Blundell’s group as a postdoc soon after its move to Birkbeck in the mid-70s, where he established a long, productive collaboration with Wood and with Jim Pitts, who now directs the PPS course. Taylor is now a professor at the University of St Andrews in Scotland where he studies the structure and mechanism of <a href="https://www.ebi.ac.uk/interpro/entry/IPR011040">sialidases</a>. These enzymes hydrolyse (break) the bond between a terminal sialic acid residue and the remainder of a polysaccharide or glycoprotein; both bacterial and viral sialidases are involved in the pathology of infectious disease. All sialidases share a catalytic domain with a characteristic <a href="http://pps16.cryst.bbk.ac.uk/course/section5/mainbeta.html">beta propeller fold</a>, but the bacterial enzymes have a separate carbohydrate-binding domain (CBD). This binds tightly to the sialic acid substrate of all sialidases, including that of influenza virus neuraminidase (which will be covered in detail in section 10 of the PPS course). Taylor and his group were awarded a grant to explore the idea that this domain, alone, might bind tightly enough to sialic acids on the surface of influenza virus host cells to prevent both virus entry and the release of progeny virions. They have now developed multi-valent CBDs that can protect mice from challenge with a lethal dose of influenza virus. Taylor suggested that, if these molecules are as successful in protecting against influenza in human trials, they might also be useful prophylactics for other respiratory pathogens that bind to cells via sialic acid receptors.
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<a href="https://www.nottingham.ac.uk/pharmacy/people/jonas.emsley">Jonas Emsley</a>, one of Wood’s many PhD students at Birkbeck, is now at the University of Nottingham where his group studies the structures and mechanisms of proteins involved in blood coagulation. His talk focused on the activation and assembly of proteases in the <a href="https://en.wikipedia.org/wiki/Coagulation#Contact_activation_pathway_.28intrinsic.29">contact system</a>, in which the presence of ‘foreign’ surfaces such as bacteria triggers several physiological processes including blood clotting. Inappropriate activation of this system has been linked to heart disease and stroke, and mice that lack either of the coagulation factors Factor XI and Factor XII are protected to some extent from thrombosis. Factor XI, which is activated by Factor XII, contains four repeats of a domain with six conserved cysteine residues that can be drawn in the shape of an apple, hence its name of ‘<a href="https://www.ebi.ac.uk/interpro/entry/IPR003609">apple domain</a>’. The protein circulates as a dimer with the monomer-monomer interactions mediated by one apple domain and the catalytic domains sitting on top of the eight apple domains like a cup on a saucer. There is a pocket on the surface of each apple domain, and the pocket on the second such domain binds a conserved tripeptide, DFP, that is found in many of its substrates. Small-molecule inhibitors of this interaction might be useful anticoagulants.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgdonUS_yADhtpz0-SSaC2Zi8h3EhgzTx7TfocT_Fj0XFB7fI7nKLkGCQu3DIyh0JxfM1tdcf8-gb6Mt0ebbN9fPNLcaTm9Jnofs6JIV6FJRjzngliykLJR-aHYwpex3cgadO12pBNR7xs/s1600/emsley-fig.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgdonUS_yADhtpz0-SSaC2Zi8h3EhgzTx7TfocT_Fj0XFB7fI7nKLkGCQu3DIyh0JxfM1tdcf8-gb6Mt0ebbN9fPNLcaTm9Jnofs6JIV6FJRjzngliykLJR-aHYwpex3cgadO12pBNR7xs/s320/emsley-fig.png" width="186" height="320" /></a>
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<b>Structure of factor XI apple domain with bound peptide substrate showing the conserved DFP motif. Image (c) Jonas Emsley</b>
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Other speakers included Birkbeck’s <a href="http://www.bbk.ac.uk/biology/our-staff/academic/helen-saibil">Helen Saibil</a>, whose ground-breaking high resolution electron microscopy of protein complexes has been covered many times in this blog (see e.g. posts from <a href="http://principlesofproteinstructure.blogspot.co.uk/2015/04/protein-machines-in-molecular-arms-race.html">April 2015</a> and <a href="http://principlesofproteinstructure.blogspot.co.uk/2013/07/groel-giving-misfolded-polypeptides.html">July 2013</a>) and <a href="https://www.crick.ac.uk/research/a-z-researchers/researchers-k-o/neil-mcdonald/">Neil McDonald</a>, now based at the <a href="https://www.crick.ac.uk/">Francis Crick Institute</a> in London, who described some largely unpublished work on the structure and mechanism of RET receptor tyrosine kinases. Appropriately, however, the final talk was devoted to Wood’s structure: SAP. It was given by <a href="http://www.port.ac.uk/school-of-biological-sciences/staff/dr-simon-kolstoe.html">Simon Kolstoe</a> who joined the Wood group in Southampton as a PhD student in 1999, moved with him back to UCL and is now at the University of Portsmouth. He first presented a ‘potted history’ of structural studies of this protein, describing how a competitive inhibitor of SAP-amyloid binding was developed as a potential treatment for amyloidosis at the turn of the millennium. This compound, <a href="https://en.wikipedia.org/wiki/CPHPC">CPHPC</a>, was found to deplete SAP levels in serum but, unfortunately, clinical amyloid levels were unchanged. A high-resolution structure of this compound binding to SAP was published in 2014 (<a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4AVV">PDB 4AVV</a>). Kolstoe and his co-workers have now turned their attention to SAP binding to DNA, which might also be clinically relevant.
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The meeting ended with the usual votes of thanks, with the award of a poster prize to Jingxu Guo from University College London, and with a gift to Wood: a molecular model of a SAP-drug complex, presented by Tony Savill of <a href="http://www.moleculardimensions.com/">Molecular Dimensions Ltd</a>.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEifCZOTv0TZ3zXyxT93L7HBMGa1jv-NnQAtAQ7FpsiMqKX0p2b-CstRRpy1y-7VRWp0qtuFq7Cm8yJ7kvA5tzkKGj_hdpIepXgAHg8uHpNI9wuII9CQ7E9L6XmyyWpXtwnahQJ4khLiy-A/s1600/Kolstoe-ActaCryst2.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEifCZOTv0TZ3zXyxT93L7HBMGa1jv-NnQAtAQ7FpsiMqKX0p2b-CstRRpy1y-7VRWp0qtuFq7Cm8yJ7kvA5tzkKGj_hdpIepXgAHg8uHpNI9wuII9CQ7E9L6XmyyWpXtwnahQJ4khLiy-A/s320/Kolstoe-ActaCryst2.png" width="320" height="320" /></a>
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<b>Image of two molecules of SAP coordinated with five molecules of CPHPC. Image (c) Simon Kolstoe, PDB 4AVV</b>
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Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-71084342583178491772016-10-10T06:12:00.000-07:002016-10-10T06:12:09.293-07:00Welcome to PPS students 2016-17!This post is extremely like those I have written at this time of year for the past few years. This is because what I have to say now is very, very similar...
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I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have just started studying Birkbeck's Principles of Protein Structure (PPS) course, and a welcome back to any who have taken a break in studies and intend to complete the course this year.
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I run this blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. I might, example, report on talks given in the ISMB seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The <a href="http://www.ismb.lon.ac.uk/Autumn2016_ISMBSeminarProgramme.pdf">programme for Autumn 2016</a> focuses on the molecular and structural biology of infectious disease; there is material on similar topics in section 10 of the PPS course, 'Protein Interactions and Function'. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas. Look out for an account of a <a href="http://bsg.crystallography.org.uk/wint16.html">conference on structural assemblies</a> at Birkbeck in December that will honour the 50-year career of one of our emeritus professors, Steve Wood.
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Some earlier posts on this blog were written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in <a href="http://principlesofproteinstructure.blogspot.co.uk/2012/03/hello-from-recent-graduate-of.html">this post</a> written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.
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Do, if you get a chance, look through some blog posts from earlier years to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!
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I particularly recommend that you look at a couple of posts from <a href="http://principlesofproteinstructure.blogspot.co.uk/2013/12/a-very-short-history-of-crystallography.html">December 2013</a> and <a href="http://principlesofproteinstructure.blogspot.co.uk/2014/07/science-week-2014-birkbeck-and-history.html">July 2014</a> about the history of structural science, particularly X-ray crystallography. Crystallography was the first method to be developed for solving the structure of biological macromolecules, and it is still the most important. The year 2014 was designated by the United Nations as the <a href="http://www.iycr2014.org/">International Year of Crystallography</a>, marking the year between the centenaries of the publication of the first papers on X-ray diffraction and the award of the <a href="http://www.nobelprize.org/nobel_prizes/physics/laureates/1915/">1915 Nobel Prize for Physics</a> to the father-and-son team of William and Lawrence Bragg who made the principal discoveries.
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So - the best of luck for the 2016-17 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our MSc in Structural Molecular Biology.
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Best wishes,
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Dr Clare Sansom
<br> Senior Associate Lecturer, Biological Sciences, Birkbeck and Tutor, Principles of Protein Structure Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com1tag:blogger.com,1999:blog-7005104906174355459.post-37157904232117712652016-09-19T10:34:00.000-07:002016-09-19T10:40:22.333-07:00Shining light on the 3D structures of membrane proteinsA symposium was held at Birkbeck on August 10, 2016 to honour one of the college’s most distinguished structural biologists, <a href="http://people.cryst.bbk.ac.uk/~ubcg25a/">Professor Bonnie Wallace</a>. This was organised by a postdoctoral member of her group, Lee Whitmore, and her long-term colleague and collaborator <a href="http://webspace.qmul.ac.uk/rwjanes/">Dr Bob Janes</a> from Queen Mary, University of London to celebrate her 65th birthday. It featured speakers from five continents, all of whom had collaborated with her or worked in her lab at some point in their careers.
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Wallace joined what was then the Department of Crystallography at Birkbeck in 1990 after holding several research positions in her native USA. Her research on the structure and function of membrane proteins has won her several prestigious awards, including the Biochemical Society’s triennial <a href="https://www.biochemistry.org/Awards/AstraZenecaAward.aspx">AstraZeneca award</a> in 2010. This is given for outstanding research in a UK or Irish laboratory that leads to the development of a new method or reagent. She has made significant contributions to both the development of <a href="http://pcddb.cryst.bbk.ac.uk/home.php">circular dichroism spectroscopy</a> as a tool for investigating the structures of proteins (including membrane proteins) at less than atomic resolution, and to studies of membrane protein structures using crystallography and electron microscopy. Her studies of <a href="https://en.wikipedia.org/wiki/Sodium_channel#Voltage-gated">voltage-gated sodium channel</a> structures have led to some important insights about their functions in health and disease.
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The symposium was divided into three sessions, with the first devoted to circular dichroism spectroscopy and the second two to membrane proteins. A <a href="http://www.bbk.ac.uk/biology/news/bbk-local?uid=979c1350f6ba476f85ff45f05442145b">general report of the day</a> has been published on the Biological Sciences website; here, to fit in with the remit of the PPS course, I concentrate on the sections on membrane proteins.
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The first talk was on electron microscopy, given, appropriately enough, by one of the pioneers of the field:<a href="http://www2.mrc-lmb.cam.ac.uk/groups/rh15/"> Richard Henderson</a> from the MRC Laboratory of Molecular Biology in Cambridge. Throughout most of the 1970s and 1980s he and his collaborator, Nigel Unwin, worked on the development of electron microscopy techniques for the study of protein structures. Most of their work involved the proton pump, bacteriorhodopsin, which is found in very high concentrations in the purple membranes of Halobacteria. At the beginning, this work was very time-consuming: it took them a year to locate the C-terminus of the protein, and another to determine the binding site of its ligand, retinal.
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The first near atomic resolution structures of this protein were obtained in the mid-1990s. At about that time, too, he switched the focus of his interest from the structures of ‘2D crystals’ of bacteriorhodopsin to those of ‘blob-like’ single particles: isolated protein chains or, more often, membrane-embedded protein complexes. The list of biologically and medically important complexes to have been solved using this technique is now growing rapidly, and includes rotary ATP synthase (see the <a href="http://principlesofproteinstructure.blogspot.co.uk/2016/08/atp-synthase-new-drug-target-for.html">previous post</a> on this blog); the next complex in the electron transport chain, known as <a href="http://">respiratory complex I</a>; and <a href="https://en.wikipedia.org/wiki/Gamma_secretase">gamma secretase</a>, which is a potential drug target for Alzheimer’s disease.
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Molecular simulation and modelling techniques have developed alongside those of structural biology and for almost as long. <a href="https://www.bioch.ox.ac.uk/aspsite/index.asp?pageid=599">Mark Sansom</a>, a professor of structural bioinformatics at the University of Oxford, described simulations of membrane proteins. He started his talk describing a program to visualise and analyse the pores through the centres of membrane proteins that was written by <a href="http://www.ebi.ac.uk/about/people/oliver-smart">Oliver Smart</a> (now at the EBI) when he was a postdoc in Wallace’s group. This program, <a href="http://www.smartsci.uk/hole/">HOLE</a>, is relatively simple but is still widely used. Sansom’s current work uses molecular dynamics to model the membrane bilayer with numbers of embedded proteins, focusing particularly on interactions between those proteins and the lipids of the membrane.
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Not surprisingly, there were several talks about the ion channels that have been a focus of so much of Wallace’s more recent research: voltage-gated sodium channels. <a href="http://hemmingslab.org/Welcome.html">Hugh Hemmings</a> from Weill Cornell Medicine, New York, USA described how these channels have become useful targets for anaesthetic drugs. General anaesthesia is a drug-induced coma characterised by unconsciousness, immobility and amnesia; an effective anaesthetic will achieve all these and a wide variety of molecules have been employed to greater or lesser effect since the nineteenth century. Many of these target proteins involved in the release of neurotransmitters by pre-synaptic nerves, including ion channels; sodium channels were first proposed as anaesthetic targets in the late 1970s but fell out of favour for several decades. Interest in this mechanism of anaesthesia has revived with the use of the bacterial proteins – a focus of Wallace’s structural studies – as a model system. Hemmings’ current studies focus on the mechanism through which volatile anaesthetics such as <a href="https://en.wikipedia.org/wiki/Isoflurane">isoflurane</a> inhibit the passage of sodium ions through these channels.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhg36SH27SLKkoIpzD2_MdlQf1ubcanHqM7ecwMYg1Uka1xlPe8GAaGSxxwUzHe47Cu5OfsorNlS-RX6OWEJGXkZTvSDR5ch_KpncXYwDVYrtr_XMNYVmehipDpvapK5MWOyusrRWQXsNg/s1600/navMS.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhg36SH27SLKkoIpzD2_MdlQf1ubcanHqM7ecwMYg1Uka1xlPe8GAaGSxxwUzHe47Cu5OfsorNlS-RX6OWEJGXkZTvSDR5ch_KpncXYwDVYrtr_XMNYVmehipDpvapK5MWOyusrRWQXsNg/s320/navMS.png" width="320" height="291" /></a></div>
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<center><i>
Crystal structure of the NavMs voltage-gated sodium channel pore depicted in ribbon motif and viewed from the membrane normal direction.
each of the four monomers in the tetrameric structure is depicted in a different colour (from <a href="http://emboj.embopress.org/content/35/8/820.long">Naylor <u>et al.</u>, 2016</a> - Wallace lab paper).
The transmembrane sodium pathway run through the middle of the structure, from top to bottom.</i></center>
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<a href="http://www.rothamsted.ac.uk/people/lfield">Lin Field</a> of Rothamsted Research, Harpenden, UK, described research leading to a very different application of sodium channel blockers: as insecticides. Insects cause an immense amount of crop damage worldwide, but non-specific insecticides might be toxic either to humans or to beneficial insects such as bees. The mechanism of the <a href="https://en.wikipedia.org/wiki/Pyrethroid">pyrethroid</a> class of insecticides was unknown when the first members of this class were patented, but they are now known to bind to voltage-gated sodium channels and prevent their closure. Structural studies of these proteins have shown how mutations that are known to lead to pyrethroid resistance can prevent the molecules from binding, and why these compounds have very little effect on the very similar mammalian channels. Researchers hope that these studies are taking us nearer to the development of ideal, ‘designer insecticides’ that are only harmful to pest species.
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Further talks were given by Wallace’s first Ph.D. student at Birkbeck, <a href="http://www.southampton.ac.uk/biosci/about/staff/dad1v12.page">Declan Doyle</a>, who is now at the University of Southampton; by <a href="https://www.sheffield.ac.uk/mbb/staff/perbullough/perbullough">Per Bullough</a> from the University of Sheffield; and by <a href="http://robinsonweb.chem.ox.ac.uk/carol-robinson.aspx">Dame Carol Robinson</a>, the first woman to be appointed as a full professor of chemistry at the University of Oxford. The symposium ended with a summary and vote of thanks from Janes, who stressed that it did not mark Wallace’s retirement: she still loves science and has many questions to answer. I hope that I will be blogging innovative research from the Wallace lab for many years to come.
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Wallace’s research has been described in this blog on several previous occasions – see in particular <a href="http://principlesofproteinstructure.blogspot.co.uk/2013/04/science-week-2013-structures-of-sodium.html">this post</a> from April 2013 and <a href="http://principlesofproteinstructure.blogspot.co.uk/2010/11/protein-structure-highlighted-in.html">this one</a> from November 2010. The use of cryo-electron microscopy to determine atomic resolution structures of proteins is covered in depth in our Techniques in Structural Molecular Biology course, which is one of the options for the second year of the Structural Molecular biology MSc.Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com1tag:blogger.com,1999:blog-7005104906174355459.post-19392508884048439212016-08-04T04:32:00.001-07:002016-08-04T04:32:52.775-07:00ATP synthase: a new drug target for tuberculosisThe London Structural Biology Club (LSBC) is a network for students and researchers working in all aspects of structural molecular biology and based in London and the south-east of England. Once a term, members get together for an afternoon of research talks and discussion followed by refreshments (generally featuring pizza and beer). These meetings are often held at Birkbeck, and we have featured them on the PPS blog before (see <a href="http://principlesofproteinstructure.blogspot.co.uk/2008/07/london-structural-biology-club-meeting.html">this post</a> from 2008 and <a href="http://principlesofproteinstructure.blogspot.co.uk/2012/08/twenty-years-of-structural-biology-in.html">this one</a> from 2012).
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The LSBC meeting for the summer term of 2016 was also held at Birkbeck, and hosted and chaired by Alfonso de Simone, a lecturer in NMR spectroscopy at <a href="http://www.imperial.ac.uk/">Imperial College London</a>. One of the four talks was given by <a href="https://www.imperial.ac.uk/people/t.meier">Thomas Meier</a>, also from Imperial College. Meier, who has worked at ETH Zurich, Switzerland, and the <a href="https://www.biophys.mpg.de/en.html">Max Planck Institute of Biophysics</a> in Frankfurt, Germany, was appointed to a chair of structural biology at Imperial just over a year ago. His research concerns the structure and function of a tiny, complex ‘molecular machine’, <a href="http://pps15.cryst.bbk.ac.uk/course/section10/atpsyn.html">ATP synthase</a> (the link is to our page on that enzyme in section 10 of the PPS course). This enzyme is a ‘rotary ATP synthase’, catalysing the conversion of the electrochemical energy of ion transfer across the cell membrane into chemical energy stored in ATP. Meier and his group have solved the complete structure of this multi-subunit enzyme complex using a combination of X-ray crystallography and electron microscopy, and shed light on its role as a target for a novel class of drugs against tuberculosis.
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ATP synthase is a highly dynamic enzyme complex, which makes it particularly difficult to study structurally. The complete enzyme comprises two motor sub-complexes; most of one, termed F<sub>o</sub>, is embedded in the membrane and the other, F<sub>1</sub>, is in the matrix. They are tightly coupled with each other and linked by subunits forming an outer stalk and an inner, central stalk. From a functional point of view, the ATP synthase consists of a rotor and stator part, both sharing subunits from the F<sub>1</sub> and the F<sub>o</sub> sectors. The motors are driven either by the proton (or sometimes Na<sup>+</sup>) motive force to form ATP, or in opposite direction by the hydrolysis of ATP to pump ions across the membrane. Similar enzymes are found in the inner membranes of bacteria and mitochondria and in the <a href="https://en.wikipedia.org/wiki/Thylakoid">thylakoid membranes</a> of chloroplasts.
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John Walker of the MRC Laboratory of Molecular Biology in Cambridge was awarded a share of the <a href="http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1997/">1997 Nobel Prize in Chemistry</a> for determining the structure of the bovine F1 motor using X-ray crystallography (e.g. PDB <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=1E79">1E79</a>). This subcomplex consists of five different types of subunits; three pairs of similar alpha and beta subunits are arranged alternately around the rotor (gamma / delta / epsilon subunits), which harbours an asymmetric coiled coil domain in the gamma subunit. The nucleotides ADP and ATP can bind to the interfaces between the beta and alpha subunits; the central gamma subunit rotates in 120<sup>o</sup> steps, causing conformational changes that in turn change the affinity of the three catalytic sites for the nucleotides. Rotation in one direction, driven by the energy of ion transfer across the membrane, leads to synthesis of ATP from ADP and phosphorus (P<sub>i</sub>); rotation in the other direction will hydrolyse ATP back into ADP and P<sub>i</sub>, thus releasing the energy required to pump ions.
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The membrane-embedded F<sub>o</sub> motor consists of the rotor part, a ring of identical c-subunits termed the c-ring and the stator part, a single-chain a-subunit that grants access and release pathways for ions. Each c-ring subunit is a helical hairpin with its N and C termini on the cytoplasmic side; the number of subunits is constant within a species but varies between species, as far as we know today, between 8 and 15 c-subunits. Meier’s group has solved the structures of a number of c-rings from different species of bacteria, helping to elucidate the rotor’s mechanism of action; essentially, protons (or in some cases Na<sup>+</sup> ions) can reach one of the c-ring subunits by an ion pathway mediated by the stator a-subunit, where they lock to a free ion binding site on the c-ring, rotate with the c-ring for almost a complete 360<sup>o</sup> turn to reach the second release pathway that leads to the other side of the membrane. The ion translocation causes rotation of the F<sub>o</sub> ring and with it the complete central stalk that protrudes the F<sub>1</sub> headpiece.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjDCrtoD9jhP9gw-Y7-UXU3Hrh9ZneKGMGaQjJrCyjbGdLt8e6i17438r5DCKucgizFkSYibAjfuSu3pLKGuNfQNBCMa0dDONEyoxbeVIslswWLvGmwi4vYrrMJYsKZ14UHa0IsBcrEtrk/s1600/trial_15_opaqueBG_5_cut_smaller_150dpi_12cm.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjDCrtoD9jhP9gw-Y7-UXU3Hrh9ZneKGMGaQjJrCyjbGdLt8e6i17438r5DCKucgizFkSYibAjfuSu3pLKGuNfQNBCMa0dDONEyoxbeVIslswWLvGmwi4vYrrMJYsKZ14UHa0IsBcrEtrk/s320/trial_15_opaqueBG_5_cut_smaller_150dpi_12cm.jpg" width="320" height="283" /></a></div>
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<center><i>Artist's impression of an ATP synthase molecule embedded in a membrane. Image © Laura Preiss, Max Planck Institute of Biophysics, Frankfurt, Germany</i></center>
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Until recently, ATP synthase has not been thought of as a drug target, principally because the structures and mechanisms of the bacterial and human enzymes are so similar. Now, however, it has emerged that it is the target for <a href="https://en.wikipedia.org/wiki/Bedaquiline">bedaquiline</a>, the first novel drug to be approved for treating <a href="http://www.who.int/mediacentre/factsheets/fs104/en/">tuberculosis (TB)</a> for over 40 years. And new drugs for TB are needed very badly: in 2015, over 9 million people contracted this disease and about 1.5 million died from it. Tens of thousands of TB cases each year are multidrug resistant (MDR) or even of the extremely drug resistant (XDR) variant for which no other clinically approved antibiotic is available anymore. Bedaquiline, however, is effective against both, MDR- and XDR-TB strains.
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Functional analysis has shown that bedaquiline, which is a diarylquinoline, acts by binding to and halting the rotation of the Fo rotor. Meier and his co-workers have now solved the structure of the drug bound to the c-ring from a similar, non-pathogenic bacterial species, <a href="https://en.wikipedia.org/wiki/Mycobacterium_phlei"><i>Mycobacterium phlei</i></a>. This structure has nine c-subunits and shares over 80% sequence identity with the <i>M. tuberculosis</i> c-subunit variant (100% match at and around the drug binding surface). The crystal structure (PDB <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4V1F">4V1F</a>; <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640650/">Preiss <i>et al.</i> (2015)</a>) shows the drug occupying the proton-binding site on each of the nine subunits and thus preventing proton transfer. This stalls the rotation of the F<sub>o</sub> motor, preventing rotation and thus the synthesis of ATP in F<sub>1</sub>. Small differences between the structures of the proton-binding sites account for the exquisite specificity of bedaquiline for the F<sub>o</sub> rings of mycobacteria and thus for its efficacy and safety as an anti-tubercular drug.
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Meier ended his talk by explaining that TB-causing bacteria will eventually – perhaps sooner rather than later – develop resistance to bedaquiline, just as they have to every previous drug that has entered the clinic. There is therefore a pressing need to develop further drugs that act at the same target, and his group’s structural studies are proving useful in the search for bedaquiline analogues.
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The London Structural Biology Club has a public Facebook group, which can be found <a href="https://www.facebook.com/groups/396037070496925/">here</a>.Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com1tag:blogger.com,1999:blog-7005104906174355459.post-39560398592702862232016-05-31T07:44:00.000-07:002016-05-31T07:44:35.539-07:00Molecules that WalkThe Department of Biological Science’s contribution to Science week 2016 kicked off on 11 April with a lecture by <a href="http://people.cryst.bbk.ac.uk/~ubcg87a/">Dr Anthony Roberts</a>, a young Principal Investigator who arrived at Birkbeck in 2014. Anthony received his B.Sc. from Imperial College in London and his Ph.D. from the University of Leeds, and spent four years as a postdoc at <a href="http://reck-peterson.ucsd.edu/">Harvard</a> in the USA before moving here to start his own research group as a Sir Henry Dale Fellow of the Wellcome Trust and Royal Society.
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Anthony began his lecture by explaining that he was going to talk about molecules that have the capacity to produce directed movement – or to ‘walk’ – and their importance for human health. These molecules are all proteins, and the context in which they move is the interior of living cells. Both the proteins he studies, kinesin and dynein, ‘walk’ on a network of highways conceptually not unlike the <a href="https://tfl.gov.uk/">transport system</a> that we use to move around London. These cellular highways are filaments called <a href="http://principlesofproteinstructure.blogspot.co.uk/2015/07/microtubules-and-microscopes-exploring.html">microtubules</a>, which, unlike our roads and railway tracks, are able to self-assemble and also to self-destruct.
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The ability to move is one of the fundamental properties of life, and scientists and philosophers have been studying it for millennia. Muscles were identified as the organs of movement in antiquity, but it was not until the mid-twentieth century that the molecules involved in muscle contraction could be identified. The Hungarian physiologist <a href="http://www.nobelprize.org/nobel_prizes/medicine/laureates/1937/szent-gyorgyi-facts.html">Albert Szent-Györgyi</a> discovered the muscle proteins now named <a href="https://en.wikipedia.org/wiki/Actin">actin</a> and <a href="https://en.wikipedia.org/wiki/Myosin">myosin</a> using very simple equipment during the Second World War.
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These proteins have similarities with kinesin and dynein, although historically they have been easier to study due to their abundance in muscle; actin forms <a href="http://pdb101.rcsb.org/motm/19">fibrils</a> and the enzyme <a href="http://pdb101.rcsb.org/motm/18">myosin</a> binds to and ‘walks’ along these filaments. This process, like all movement, requires energy, and this is obtained from the cell’s power source, the small molecule adenosine triphosphate (ATP). The part of the myosin molecule that binds to actin, which is called its head, breaks a phosphate bond in this molecule to liberate energy and power the walking motion; many of these ‘power strokes’ together cause the muscle fibre to contract.
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Ideally, we would want to watch this, or any other form of molecular motion, in real time, but this is impossible because molecules are far too small: smaller than the wavelength of light, so they cannot be viewed in a light microscope. Studies of molecular structure require techniques like X-ray crystallography and electron microscopy, both of which have been used to study motor molecules.
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However, neither of these techniques can do more than generate still images. Movement can only be inferred by taking lots of snapshots of the molecules at different points during the movement cycle, rather like the earliest movies. We have now built up a complete picture of actin and myosin that is detailed enough for the positions of individual atoms to be seen clearly.
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Not all movement in nature, however, uses muscles. Single-celled organisms – the ‘animalcules’ observed by pioneer microscopist <a href="https://en.wikipedia.org/wiki/Antonie_van_Leeuwenhoek">Antonie van Leeuwenhoek</a> in the 1670s – have directed movement, as do bacteria, and these have neither muscles nor nervous systems. And directed movement also occurs inside cells. A good example of this is the division of replicated DNA between daughter cells during cell division.
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The interior of all cells is a viscous mixture, crowded with molecules; it is possible for small molecules to move from one part of a cell to another through diffusion, but this process would be impossibly slow for larger ones. Motor proteins, on the other hand, can carry ‘cargo’ molecules across cells remarkably quickly and efficiently. Motor proteins can traverse a distance of 0.1 mm – the length of a large animal cell – in two minutes, which in terms of lengths per second is approximately three times faster than a car.
<p>
Both the motor proteins studied in Anthony’s lab, kinesin and dynein, ‘walk’ along microtubules inside cells. These filaments typically form with one end towards the centre of the cell, and its nucleus, and the other towards the cell periphery, and the motor proteins move in opposite directions: dynein towards the nucleus, and kinesin towards the cell edge.
<p>
Any kind of directed movement by molecules is challenging for several reasons. Motor molecules have no equivalent of our nervous systems for controlling movement, and they are far too small to be held on their tracks by gravity; instead, they grip the microtubules using chemical forces. They experience negligible inertia, and are constantly buffeted by other molecules in the cell. It would therefore be catastrophic for the whole of a walking molecule to leave its path at once.
<p>
The structure and function of conventional <a href="http://pdb101.rcsb.org/motm/64">kinesin</a> are now fairly well understood. It consists of two identical protein chains, and each chain has two major domains separated by a short linker. The larger domain of each chain coils together to form a single long stalk; the smaller domain is globular and attaches to the microtubule, so the molecule looks rather like a single leg with two feet. Each of the feet is an enzyme that generates the energy for the motion by breaking down ATP to form ADP and release a phosphate group, and it cycles between ATP-bound, ADP-bound and empty states.
<p>
The step between ADP-bound and empty is a bottleneck that can be relieved when the foot attaches to the microtubule in a particular position, ensuring that the whole molecule moves in the correct direction. The trailing foot is released from the microtubule and the cycle begins again once ATP has bound to the front foot, triggering a conformational change in the whole molecule.
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The core of kinesin is similar in structure to myosin, suggesting that these two proteins have a common ancestor. The other microtubule-bound motor protein, dynein, has a different origin. Although we still know comparatively little about it, it was actually the first of the microtubule-bound motor proteins to be discovered: this was in the 1960s, when it was found as the protein that generates the force that allows protozoa and sperm cells to swim. Anthony’s group, however, has been studying how it functions inside cells to move ‘cargo’ – often nucleic acids or other proteins – from the edges of the cell towards its interior. It also helps to pull the duplicated genetic material between the two halves of the cell during cell division.
<p>
Dynein is a much larger and more complex molecule than the other motor proteins. Its structure, like those other proteins, has several components: in this case, a stalk, a ring and a tail, with a linker between the stalk and the ring. Much of what we know about this large structure has come from electron microscopy, and more recently X-ray crystallography.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgp88EPW8JKnqE2k8KPnQ6Y808jQGGBy0GRsAKU54FmEGUFR-PjUzhblMWnV0qZt66kYbBzZpBg8IIWq7Oux8OeJiS9uOBhwv5Yp3CWu9RlVDd-VyZYwNTnlgPQ1rry60-Fku58zaGf6DI/s1600/dynein.jpeg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgp88EPW8JKnqE2k8KPnQ6Y808jQGGBy0GRsAKU54FmEGUFR-PjUzhblMWnV0qZt66kYbBzZpBg8IIWq7Oux8OeJiS9uOBhwv5Yp3CWu9RlVDd-VyZYwNTnlgPQ1rry60-Fku58zaGf6DI/s320/dynein.jpeg" /></a></div>
<br>
<center><b>The structure of dynein; the stalk is shown in yellow and the linker in magenta.</b></center>
<p>
Anthony’s group and others have developed a model in which the main mechanical element is the linker, which bends and straightens to displace the cargo-bound end of the structure along the microtubule in the direction of travel. The image shown here is a still from an <a href="http://people.cryst.bbk.ac.uk/~ubcg87a/animation.html">animated model</a> of how dynein generates movement, which remains speculative in places and is helping to stimulate new experiments in these areas. It is also incomplete, as it only shows one half of the molecule: we do know that dynein, like kinesin, is a biped, but exactly how its ‘feet’ are coordinated remains at the frontier of our knowledge.
<p>
Anthony ended his talk by discussing some actual and potential medical applications of studies of walking molecules. Some commonly used anti-cancer drugs, including <a href="https://en.wikipedia.org/wiki/Paclitaxel">taxol</a>, work by stabilising microtubules to prevent motion and therefore stop cancer cells from dividing. Molecules that interact with motor proteins are also being studied as potential treatments for neurodegenerative diseases and for some types of heart disease. One such compound is a myosin activator, omecamtiv mecarbil, which is showing promise as a treatment for heart failure. And we are likely to discover further applications as we learn more about these fascinating walking molecules.Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-54520615838583019572016-05-10T06:52:00.001-07:002016-05-10T06:52:30.005-07:00Crystallography: from Chocolate to Drug DiscoveryBirkbeck has already established lecture series in honour of some of its most distinguished alumni. Until 2016, however, <a href="https://en.wikipedia.org/wiki/Rosalind_Franklin">Rosalind Franklin</a> – co-discoverer of the DNA structure and perhaps the most widely recognisable of its ‘famous names’ – was missing from the list of honourees. This gap has now been filled; the annual Rosalind Franklin lecture forms part of the college’s <a href="http://www.ecu.ac.uk/equality-charters/athena-swan/">Athena SWAN</a> programme and will always be given by a distinguished woman scientist. And fittingly, the inaugural lecture, which was part of <a href="http://www.bbk.ac.uk/science/about-us/events/science-week/">Science Week 2016</a>, was devoted to Rosalind Franklin’s own discipline, crystallography. <a href="http://www.bioch.ox.ac.uk/aspsite/index.asp?sectionid=garman">Elspeth Garman</a>, Professor of Molecular Biophysics at Oxford University, gave an entertaining and illuminating lecture to a large audience that included Rosalind’s sister, the author Jenifer Glynn.
<p>
Garman began her lecture by showing a short <a href="http://www.oxfordsparks.ox.ac.uk/content/case-crystal-clarity-0">video</a> that she had produced for OxfordSparks.net that used a ‘little green man’ to illustrate the method of X-ray crystallography that is used to obtain molecular structures from crystals. The rest of the lecture, she said, would simply go through that process more slowly. She started by showing some beautiful examples of crystals. All crystals are formed from ordered arrays of molecules. They can be enormous, such as crystals of the mineral selenite in a cave in Mexico that measure <a href="https://en.wikipedia.org/wiki/Cave_of_the_Crystals">over 30’ long</a> or too small to be visible with the naked eye.
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In the early decades of crystallography, structures could only be obtained from crystals of the smallest, simplest molecules: the first structure of all, published in 1913 by the father-and-son team of W.H. and W.L. Bragg, was of table salt. When they were jointly awarded the <a href="http://www.nobelprize.org/nobel_prizes/physics/laureates/1915/">Nobel Prize for Physics in 1915</a>, the younger Bragg was a 25-year-old officer in the trenches on the Western Front. His record as the youngest Nobel Laureate was unbroken until Malala Yousafzai’s Peace Prize in 2014.
<p>
The Braggs’ discoveries paved the way for studies of the structures of many, many substances: including the chocolate of the lecture title. Few of the audience can have known that chocolate exists in six different crystal forms, or that only one of these (Form V) is good to eat. The process of ‘tempering’ – a series of heating and cooling steps – is used to ensure that it solidifies in the correct form.
<p>
Garman then moved on to talk about her own field of protein crystallography. Proteins are the ‘active’ molecules in physiology, and they are formed from long, linear strings of 20 different ‘beads’ (actually, small organic molecules known as amino acids). Chemists can quite easily find out the sequence of these beads in a protein, but it is impossible to work out from this the way that the string will fold up into a definite structure ‘like a piece of wet spaghetti’. And it is this structure that places different units with different chemical properties on the surface or in the interior of the protein, or near each other, and that therefore determines what the protein will do.
<p>
Protein crystallography only became technically possible in the mid-twentieth century, and even then it was a painfully slow and complex process that could only be used to study the smallest, simplest proteins. <a href="https://en.wikipedia.org/wiki/Dorothy_Hodgkin">Dorothy Hodgkin</a>, also a professor at Oxford, won her Nobel Prize in Chemistry in 1964 for the structures of two biologically important but fairly small molecules: penicillin, with 25 non-hydrogen atoms and vitamin B12, with 80. She is perhaps better known for solving the structure of insulin, the protein that is missing or malfunctioning in diabetics. This has 829 non-hydrogen atoms; in contrast, the 2009 Chemistry Nobel Prize was awarded for the structure of the <a href="https://en.wikipedia.org/wiki/Ribosome">ribosome</a>, the large (by molecular standards) ‘molecular machine’ that synthesises proteins from a nucleic acid template. The bacterial ribosome used for the Nobel-winning structural studies is well over 300 times larger than insulin, with over a quarter of a million atoms.
<p>
Protein structures are not only beautiful to look at and fascinating to study, but they can be useful, particularly for drug discovery. Many useful drugs have already been designed at least partly by looking at a protein structure and working out the kinds of molecule that would bind tightly to it, perhaps blocking its activity. Some viral proteins have been particularly amenable to this approach. Rosalind Franklin did some of the first research into virus structure when she was based at Birkbeck, towards the end of her tragically short life, and her student <a href="https://en.wikipedia.org/wiki/Aaron_Klug">Aaron Klug</a> cited her inspiration in his own <a href="http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1982/klug-lecture.html">Nobel lecture in 1982</a>. X-ray crystal structures were used in the design of the anti-flu drugs Relenza™ and Tamiflu™ and of HIV protease inhibitors, and more recently still structures of the foot and mouth virus are helping scientists develop new vaccines for tackling this potentially devastating animal disease. The foot-and-mouth virus structure even made the front page of the <i>Daily Express</i>.
<p>
The equipment that Dorothy Hodgkin and her contemporaries used to solve protein structures in the 1960s and 1970s looks primitive today. Now, almost every step of protein crystallography has been automated. Powerful beams of X-rays generated by synchrotron radiation sources, such as the UK’s <a href="http://www.diamond.ac.uk/Home.html">Diamond Light Source</a> in Oxfordshire, allow structures to be determined quickly from the smallest crystals. It is even possible to control some of these machines remotely; Garman has operated the one at Grenoble from her sitting room. Yet there is one step that has changed remarkably little. It is still almost as difficult to get proteins to crystallise as it was in the early decades. Researchers have to select which of a large number of combinations of conditions (temperature, pH and many others) will persuade a protein to form viable crystals. Guesswork still plays a large part and some researchers seem to be ‘better’ at this than others: Garman adds the acronym ‘GMN’ or ‘Grandmother’s maiden name’ to her list of conditions to reflect this.
<p>
Yet, with every step other than crystallisation speeded up and automated beyond recognition, the trickle of new structures in the 70s and even 80s has become a torrent. Publicly available structures are stored online in the <a href="http://www.rcsb.org/pdb/home/home.do">Protein Data Bank</a>, which started in 1976 with about a dozen structures: it now (May 2016) holds over 118,000. Protein crystallography as a discipline is thriving, but there are many challenges ahead. We are only now beginning to tackle the 70% or so of human proteins that are only stable when embedded in fatty cell membranes and are therefore insoluble in water. It is possible to imagine a time when it is possible to solve the structure of a single molecule, with no more need for time-consuming crystallisation. And, hopefully, women scientists will play at least as important a role in the second century of crystallography as they – from Quaker Kathleen Lonsdale, who developed important equations while jailed for conscientious objection during World War II, through Franklin and Hodgkin to Garman and her contemporaries – have in the first. Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-41720337050553695192016-03-09T02:40:00.000-08:002016-03-09T02:40:10.102-08:00Evolution and Assembly of Protein ComplexesSince 2003, all Birkbeck researchers in structural biology and allied disciplines have collaborated with colleagues at UCL in the <a href="http://www.ismb.lon.ac.uk/">Institute of Structural and Molecular Biology</a>. The Institute holds a varied series of events throughout the year, including a programme of research seminars arranged termly around current themes in molecular and structural biology research. The theme for the spring term 2016 seminar programme has been ‘<a href="http://www.ismb.lon.ac.uk/ISMBSeminarsSpring2016-ProteinDynamics.pdf">Protein Dynamics: from Folding to Function</a>’; one of the first of the distinguished scientists invited to present their research under that theme was <a href="http://www.ebi.ac.uk/research/teichmann">Sarah Teichmann</a> from the EMBL – European Bioinformatics Institute and the Sanger Institute at Hinxton, near Cambridge, UK. She gave a fascinating talk that linked evolution and protein folding to the topic of Section 7 of the PPS course, <a href="http://pps15.cryst.bbk.ac.uk/course/section7/index.html">quaternary structure</a> (or the assembly of protein complexes).
<p>
Teichmann has won many awards in what is still quite a short research career, including the Biochemical Society’s <a href="https://www.biochemistry.org/Awards/TheColworthMedal.aspx">Colworth Medal</a> for ‘an outstanding research biochemist under the age of 35’ (2011) and the EMBO Gold Medal (2015). Last year she was elected a Fellow of the prestigious <a href="http://www.acmedsci.ac.uk/">Academy of Medical Sciences</a>, with a citation that commended her as representing ‘a new breed of scientists at the interface between computational and experimental molecular biology’. She is also an advocate for women in science and has written a children’s novel.
<p>
She began her seminar by asking two related questions: ‘how do protein complexes assemble?’ and ‘how do protein complexes evolve?’ and by misquoting the poet <a href="https://en.wikipedia.org/wiki/John_Donne">John Donne</a>: ‘no protein [man] is an island’. Many proteins are functional only when bound to others to form complexes, and in the crowded environment of a cell each newly synthesised protein has only a limited amount of time to find its partners and form a stable complex. Much can be learned about the evolution and dynamics of complex formation by studying the complexes that are available in the <a href="http://www.rcsb.org/pdb/home/home.do">Protein Data Bank</a>. Her group’s evaluation of these structures has contributed to the software that the PDB uses to predict the functional biological unit (monomer, dimer or multimer) for each structure in the PDB, and has led to the <a href="http://www.3dcomplex.org/">3Dcomplex.org</a> database of protein complexes. This database provides a hierarchical classification of now over 30,000 protein quaternary structures. Each complex is represented using <a href="https://en.wikipedia.org/wiki/Graph_theory">graph theory</a> as a simple 2D figure or ‘mini-graph’, with each polypeptide chain as a node and each interaction surface between two chains as an edge. These little graphs make it easier to distinguish between topologies involving the same number of subunits: for example, a complex of six identical protein chains may be a simple hexamer with <a href="http://pps15.cryst.bbk.ac.uk/course/section7/cyclic2.html">6-fold rotational symmetry</a> (such as the traffic ATPase [PDB 1g6o]) or a dimer of trimers with <a href="http://pps15.cryst.bbk.ac.uk/course/section7/dihedral2.html">32 symmetry</a> (such as annexin XII [PDB 1aei]). The links here are to the pages describing those proteins in Section 7 of the PPS course material.
<p>
Alongside the hierarchy described in the 3Dcomplex database, protein complexes can be divided into two large groups: homomers, which consist of multiple copies of the same polypeptide chain, and heteromers involving different chains. (Haemoglobin, a tetramer with two alpha and two closely related beta chains, is arguably an intermediate between the main two types.) Teichmann spent the rest of her lecture addressing three related questions about the assembly of both homomers and heteromers:
<p>
i) Does the assembly of protein complexes drive evolution? <br>
ii) What are the mutational mechanisms involved in complex formation? <br>
iii) Can the principles of protein assembly be used to predict topologies that have not yet been seen? <br>
<p>
Starting with the first question, from an evolutionary point of view the simplest complex to form is a homodimer with two copies of the same monomer; one mutation that turns part of a protein surface into a ‘sticky patch’ is all that is necessary to stabilise dimer formation. Not surprisingly, the homodimer is also the commonest type of quaternary structure found in the PDB. Once a protein has dimerised, additional monomers can be added to form larger complexes with cyclic symmetry, or the dimer itself can (for example) dimerise. The order in which the interfaces in a multimer formed during evolution can be predicted from the amount of surface area buried by the formation of each interface, with the largest surface areas being buried first. This simple rule applies to complicated assemblies as much as to simple ones, and to heteromers as much as to homomers. Therefore, for all but the very simplest structures, it is almost impossible to predict the form that a complex will take unless you know the order in which the subunits assemble. <a href="http://www.hgu.mrc.ac.uk/people/j.marsh.html">Joseph Marsh</a>, a former postdoc in Teichmann’s group now working at the MRC Human Genetics Unit in Edinburgh, represents this here in an analogy with the assembly of flat-pack furniture, with and without instructions.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiDGOTfdCX_8Tibmnfrvh5KbEoKn1qabxTQyNAyIa_CPaVdwzsodkyqx5PRH9t3wtQohI_xI2asg7-_3l1nvV5iYWIorPTE1ypXEvCqgxAshddgMXVsH-pQ8DpOpdTmhGL0SXE3IGS5JE8/s1600/ikea_assembly_joe.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiDGOTfdCX_8Tibmnfrvh5KbEoKn1qabxTQyNAyIa_CPaVdwzsodkyqx5PRH9t3wtQohI_xI2asg7-_3l1nvV5iYWIorPTE1ypXEvCqgxAshddgMXVsH-pQ8DpOpdTmhGL0SXE3IGS5JE8/s320/ikea_assembly_joe.jpg" /></a></div>
<center><i>Illustration © Joseph Marsh, MRC Human Genetics Edinburgh</i></center>
<p>
Teichmann tested some of her predictions of protein assembly pathways using mass spectrometry in collaboration with <a href="http://robinsonweb.chem.ox.ac.uk/carol-robinson.aspx">Professor Dame Carol Robinson</a>’s group at the University of Oxford, and found that seven out of nine pathways and 22 out of 27 steps within those pathways had been predicted correctly. This hierarchy of subunit assembly can also be used to predict the evolution of a complex, so it is clear that the assembly of protein complexes can indeed drive evolution.
<p>
Turning to the second question, Teichmann used specific examples of protein families that take up different quaternary structures in different species, including the PyR family of bacterial pyrimidine operon attenuators, to explore the evolutionary mechanisms that take a protein from one that is most stable as a monomer to different multimeric forms. These can involve direct mutations at the interface between subunits (for example, making the protein surface ‘stickier’ or creating a salt bridge) and other so-called ‘allosteric’ mutations that change the protein structure to allow different interfaces to form. Often, the difference between (for example) a protein that is stable as a dimer and one that is stable as a tetramer will come down to changes in a few amino acids. In the case of the PyR attenuator family, mutations away from the interface drive a conformational change that is equivalent to the one that occurs when the protein binds DNA, and so stabilise multimer formation.
<p>
Finally, Teichmann considered the use of the assembly principles that she had outlined in predicting the form that a protein complex would take from scratch. Most basic steps in complex assembly, as described earlier, can be grouped into one of three categories: dimerization of one or more chains, adding an identical subunit or subunits to a complex (cyclization) and adding a different type of subunit. These can be combined in different ways to form a large number of possible quaternary structure topologies. So far, about 120 different topologies are represented in the PDB, with four or five new ones being added each year, and the vast majority of these fit into one of Teichmann’s topologies. She assembled all the predicted topologies, including those not yet observed, into a ‘periodic table of protein complexes’ (<a href="http://science.sciencemag.org/content/350/6266/aaa2245.short">S.E. Ahnert <i>et al</i>., <i>Science</i> <b>350</b>, aaa2245 (2015)</a>). This table has already been seen to correctly predict the topology of some newly determined complexes that were not included in the original list. Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-3406614760175097712015-11-19T08:31:00.000-08:002015-11-19T08:31:15.781-08:00Bernal Lecture 2015: Terminating Protein SynthesisProfessor <a href="http://https://en.wikipedia.org/wiki/John_Desmond_Bernal">J.D. Bernal</a>, known to his colleagues and contemporaries as ‘Sage’, spent much of his career as a professor of Physics at Birkbeck and became the first chair of the Department of Crystallography in 1963. When he retired in 1968 the college founded a series of lectures in his honour. The first of the Bernal Lectures, in 1969, was given by <a href="https://en.wikipedia.org/wiki/Dorothy_Hodgkin">Dorothy Hodgkin</a>, winner of the 1964 Nobel Prize for Chemistry for solving the structures of ‘important biological substances’, mainly penicillin and vitamin B12. In 2015 we were honoured to welcome another Chemistry Nobel Laureate to give this annual lecture. <a href="https://en.wikipedia.org/wiki/Venkatraman_Ramakrishnan">Professor Sir Venki Ramakrishnan</a> of the MRC Laboratory of Molecular Biology in Cambridge was <a href="http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2009/">awarded the prize in 2009</a> with Thomas Steitz of Yale University, USA and Ada Yonath of the Weizmann Institute of Science, Israel, for studies of the structure and function of the ribosome: the ‘molecular machine’ that catalyses the synthesis of proteins from their messenger RNA (mRNA) templates.
<p>
The lecture, held on 19 October 2015, was introduced by David Latchman, Master of Birkbeck College and Professor of Genetics. He welcomed three generations of Bernal’s descendants to Birkbeck, highlighted the success of the lecture series in attracting some of the most distinguished researchers in structural biology and allied disciplines, and explained that the topic of the lecture overlapped with some of his own research interests in the regulation of gene expression.
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Prof. Ramakrishnan began his lecture by explaining that protein synthesis was a complex process, involving many proteins as well as the ribosome itself, and that he would be talking about a particular point in this process: namely, how it ends (formally, the termination of protein translation). He showed an image of a ribosome in the process of protein synthesis that, he explained, represented the culmination of 40 years’ work on its structure, and explained how the linear mRNA molecule wound through a cleft between the two subunits of the ribosome. As the mRNA passes through the ribosome each of its three-base ‘codons’ comes into contact with three sub-parts of the ribosome’s active site – the A-site, P-site and E-site – in turn. When a codon enters the A-site it binds to the anti-codon of the transfer RNA (tRNA) carrying the next amino acid; the amino acid is bonded to the previous amino acid in the growing protein chain in the P-site, and the now empty tRNA released from the ribosome in the E-site. This continues until the new protein chain is complete. This is signalled by one of the so-called ‘stop codons’ UGA, UAG and UAA, which have no corresponding tRNAs, entering the ribosome’s A-site. The new protein is released from the ribosome to fold into its native structure, and the ribosome subunits dissociate.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg8mYXN4c_zgblOtN3t8PsVwb7DvxOTf9TMyWgESag1knQnsu3U1tZxjSez75argQHGtZTOAcwYvS700rpxadKpQLuKKziLQgNAQkyCrqy6VGv5aaPIM-HmdBPaq4QjWa8JcuxfVYsNxAk/s1600/121-70SRibosomes_elongation.tif" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg8mYXN4c_zgblOtN3t8PsVwb7DvxOTf9TMyWgESag1knQnsu3U1tZxjSez75argQHGtZTOAcwYvS700rpxadKpQLuKKziLQgNAQkyCrqy6VGv5aaPIM-HmdBPaq4QjWa8JcuxfVYsNxAk/s320/121-70SRibosomes_elongation.tif" /></a></div>
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<center>
<b>Diagram of a ribosome showing the three tRNA binding sites during protein elongation <br>
Taken from the PDB ‘<a href="http://http://www.rcsb.org/pdb/101/motm.do?momID=121">Structural View of Biology: The Ribosome</a>’ <br>
© David Goodsell, 2010</b>
</center>
<p>
Although the process of adding amino acids to a protein chain is extremely similar in all organisms, there are significant differences between bacteria (prokaryotes) and eukaryotes in the process of termination – as, indeed, there are in the initiation of protein synthesis. We are beginning to understand these mechanisms only now that we can obtain high resolution structures of ‘snapshots’ of the ribosome at different points during the protein synthesis cycle and follow the sequence of conformational changes that occur then.
<p>
All stop codons are recognised and decoded by proteins known as a release factors. Bacteria have two of these: RF1 recognises UAG, RF2 recognises UGA and they both recognise UAA. Eukaryotes have only one RF, which can recognise all these codons. These three proteins all have a common sequence motif, GGQ, which is known to be involved in the release of the protein from the ribosome. The structures of the eukaryotic and prokaryotic release factors are different, but all bind to the ribosome in such a way that the GGQ motif and the part of the structure that recognises the stop codon are exactly the same distance apart as the length of a tRNA molecule. These parts of the protein will therefore interact with the peptide and the stop codon at the same time.
<p>
Prof. Ramakrishnan and his group spent years trying to obtain near atomic resolution structures of functional ribosome-release factor complexes; this problem was solved initially for the smaller prokaryotic ribosomes but now for eukaryotic ones as well. In all cases, the release factors bind to the ribosome in a different way to the tRNA molecules, inducing a different conformational change in the complex. Using the bacterial structures, the group was able to understand why the factors RF1 and RF2 only recognise the codons that they do.
<p>
In the case of eukaryotes, not only were the structures harder to obtain, but the basic question to be asked was more complex: how can a single protein recognise the stop codons UGA, UAG and UAA, but not UGG (<a href="http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Codons.html">which codes for the amino acid tryptophan</a>)? Ramakrishnan reasoned that mutating the GGQ motif in the release factor would make it inactive, and that binding this mutant protein to the ribosome with an ATPase might ‘trap’ the complex in the structure that it takes up before protein is released and allow the structure to be determined. Electron micrographs of these structures have shown that the three anti-codons and no others are recognised through a combination of base stacking and hydrogen bonding. Ramakrishnan ended his talk by comparing anti-codon binding to a <a href="https://en.wikipedia.org/wiki/NAND_gate">NAND gate</a> in electronics, with G representing ‘1’ and A ‘0’: any combination except GG (‘11’) in the second and third anti-codon positions leads to termination of translation and protein release.
<p>
This work was published in <i><a href="http://www.nature.com/nature/journal/v524/n7566/full/nature14896.html">Nature</a></i> in August 2015 and the (very large!) structures of the complexes – one snapshot with the release factor bound in each of the ribosome subsites – are available in the PDB as entries <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=3JAG">3JAG</a>,<a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=3JAH">3JAH</a> and <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=3JAI">3JAI</a>. These are some of the most recent of the 103 PDB structures on which Prof Ramakrishnan has so far been named as an author; you can view them all on a <a href="http://www.rcsb.org/pdb/author/Ramakrishnan%2C+V.">timeline</a> on the PDB site.
<p>
<br>
<i>
<u>Note:</u> If you are reading this blog post as a current PPS student, don’t be surprised if you find it difficult to understand. We will cover the structure and mechanism of the ribosome later in the course (in Section 8: The Protein Lifecycle). If you bookmark this blog post and come back to it after you have studied that section you should find that you can make much more out of it.
</i>
Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-48871435431186317082015-09-29T09:40:00.001-07:002015-09-29T09:40:55.345-07:00Welcome to PPS students 2015-16!This post is extremely like those I have written at this time of year for the past few years. This is because what I have to say now is very, very similar...
<p>
I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have just started studying Birkbeck's Principles of Protein Structure (PPS) course, and a welcome back to any who have taken a break in studies and intend to complete the course this year.
<p>
I run this blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. I might, example, report on talks given in the ISMB seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The <a href="http://www.ismb.lon.ac.uk/ISMBSeminarsAutumn2015_TranscriptionInContext.pdf">programme for Autumn 2015</a> focuses on transcription, which is the process through which DNA is copied into RNA; this will be covered in some detail in one of the later sections of the course, the Protein Lifecycle. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas. Look out for an account of a lecture to be given at Birkbeck in October by <a href="http://www2.mrc-lmb.cam.ac.uk/group-leaders/n-to-s/venki-ramakrishnan/">Venki Ramakrishnan</a>, who was awarded a share of the <a href="http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2009/">Nobel Prize for Chemistry</a> in 2009 for structural studies of ribosomes and will shortly take over as President of the <a href="https://royalsociety.org/">Royal Society</a>.
<p>
Some posts on this blog are written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in <a href="http://principlesofproteinstructure.blogspot.co.uk/2012/03/hello-from-recent-graduate-of.html">this post</a> written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.
<p>
Do, if you get a chance, look through some blog posts from earlier years to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!
<p>
I particularly recommend that you look at a couple of posts from <a href="http://principlesofproteinstructure.blogspot.co.uk/2013/12/a-very-short-history-of-crystallography.html">December 2013</a> and <a href="http://principlesofproteinstructure.blogspot.co.uk/2014/07/science-week-2014-birkbeck-and-history.html">July 2014</a> about the history of structural science, particularly X-ray crystallography. Crystallography was the first method to be developed for solving the structure of biological macromolecules, and it is still the most important. The year 2014 was designated by the United Nations as the <a href="http://www.iycr2014.org/">International Year of Crystallography</a>, marking the year between the centenaries of the publication of the first papers on X-ray diffraction and the award of the 1915 Nobel Prize for Physics to the father-and-son team of William and Lawrence Bragg who made the principal discoveries.
<p>
So - the best of luck for the 2015-16 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our MSc in Structural Molecular Biology.
<p>
Best wishes,
<p>
Dr Clare Sansom Senior Associate Lecturer, Biological Sciences, Birkbeck and Tutor, Principles of Protein Structure Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-74098984134834595852015-07-08T07:53:00.001-07:002015-07-08T07:53:35.024-07:00Microtubules and Microscopes: Exploring the CytoskeletonElectron microscopist <a href="http://www.bbk.ac.uk/biology/our-staff/academic/carolyn-moores">Carolyn Moores</a>, the most recently appointed professor in the Department of Biological Sciences at Birkbeck, gave her inaugural lecture at the college on June 1.
<p>
Moores arrived at Birkbeck in 2004 to start her research group and has risen rapidly and steadily up the academic ladder ever since. Introducing the lecture, the Master of Birkbeck, David Latchman, explained that Moores’ CV stood out in every way; she was clearly as gifted a teacher and administrator as she was a researcher. Furthermore, as she has won several awards for science communication, he predicted that the audience would be in for a treat. We were not disappointed.
<p>
Moores began her lecture by saying that she would talk about three different things: her own career development; her group’s research into the structure and function of microtubules; and the advancement of women in science, a cause that is close to her heart.
<p>
She remembered that she had wanted to work as a scientist as soon as she knew what a laboratory was, and she started young, as an intern in a research lab at Middlesex Hospital while still in the sixth form. School was followed by a BSc in Biochemistry at Oxford and a PhD in <a href="http://www2.mrc-lmb.cam.ac.uk/group-leaders/emeritus/john-kendrick-jones/">John Kendrick-Jones’ lab</a> at the world-famous Laboratory for Molecular Biology (LMB) in Cambridge. She then moved to work as a post-doc with <a href="http://www.scripps.edu/milligan/">Ron Milligan</a> at the Scripps Research Institute in La Jolla, California, USA, and it was there that she began her studies on microtubules.
<p>
The award of a David Phillips research fellowship in 2004 gave her the opportunity to return to the UK as an independent researcher. She explained that there were three reasons – or more accurately three people – that led her to choose to come to Birkbeck. Working in electron microscopy, she was inspired by the work of <a href="http://www.bbk.ac.uk/biology/our-staff/academic/helen-saibil">Helen Saibil</a>, one of the UK’s principal exponents of that technique; she had known <a href="http://www.bbk.ac.uk/biology/our-staff/academic/professor-nicholas-keep">Nicholas Keep</a>, then a lecturer in Biological Sciences, as a friend since her time at the LMB; and she knew that she would value the interdisciplinary working environment of the <a href="http://www.ismb.lon.ac.uk/">Institute for Structural Molecular Biology</a> under the ‘inspired’ leadership of <a href="http://www.bbk.ac.uk/biology/our-staff/academic/gabriel-waksman">Gabriel Waksman</a>.
<p>
Moores then moved on to discuss the main topic of her group’s research: the three-dimensional structure, function and role in disease of tiny cylindrical structures known as <a href="https://en.wikipedia.org/?title=Microtubule">microtubules</a>. These are one of the building blocks of the cytoskeleton, which forms a framework for our cells in the same way that our skeletons form a framework for our bodies. They are about 25nm in diameter, which puts them firmly into the ‘nano-scale’ of biology that is easily studied using electron microscopy.
<p>
There is a cytoskeleton in every living cell, and it, and the microtubules that form it, are involved in many important cellular processes including shape definition, movement and cell division. Diseases as diverse as cancer, epilepsy, neurodegeneration and kidney disease have been linked to microtubule defects. Understanding their fundamental structure and function, as Moores’ group
aims to, should help in understanding these disease processes and perhaps also in developing effective treatments.
<p>
Microtubules are built up from many copies of a small protein called <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=1TUB">tubulin</a>, which, in turn, is a dimer of two similar proteins called alpha and beta tubulin. These tubulin dimers make contacts with each other both head-to-tail and side-to-side to create the cylindrical microtubule wall, fuelled by energy derived from the molecule GTP. Each tubulin unit has a definite “top” and a “bottom” and, as the units are oriented in parallel, so has the complete microtubule.
<p>
Microtubules are dynamic structures; they continue to grow by the addition of tubulin units to one end as long as GTP is available, and then begin to unravel and shrink. This dynamism, which allows them to respond to the changing needs of the cell, is essential for their function in healthy cells. In particular, microtubules organise chromosome structures during <a href="https://en.wikipedia.org/wiki/Mitosis">cell division</a> and are therefore necessary for cell proliferation. As cancer is a disease of uncontrolled cell proliferation, it is possible to imagine that a molecule that could specifically block microtubule growth and assembly in the nucleus might be useful as an anti-cancer drug.
<p>
Moores and her group are aiming to understand the process of microtubule growth at as high resolution as possible, using electron microscopy. Unfortunately, however, the most detailed images can only be obtained if the specimen is at very low temperatures (in so-called cryo-elecron microscopy) and using this means that the dynamics of the specimens must be “frozen” into a still image. While it is now possible to see the individual tubulin subunits in the static microtubule images, many details of their structure can only be inferred from computational analysis.
<p>
Moores went on to describe one project in her lab in a little more detail. This was an investigation of the structure and role of proteins that bind only to growing microtubule ends, falling off when the growth stops. It is possible to obtain low-resolution images of microtubules in which these molecules have been made to fluoresce, so only growing microtubules are tracked.
<p>
In order to understand the growth process in detail, the group developed an analogue of the GTP “fuel” molecule which can bind to the tip of a microtubule that is extending but not break down to release its energy, so the microtubule does not in fact grow. This forms a static analogue of a growing microtubule that retains all the characteristics of the dynamic structure but that can be studied at low temperatures.
<p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjFo_acnborsnXIpqIgSiA0zXX3f_UQh48RbrI5gacrBQLsygqKwBg3o6lO_m-m1FqHyVLfJjof2EFwBW4xOauNXHbGYnyEnisMkt3cKsNe2aNIw1lrg-CrgVrGW2IivyySWF5sov1HG0s/s1600/EB1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjFo_acnborsnXIpqIgSiA0zXX3f_UQh48RbrI5gacrBQLsygqKwBg3o6lO_m-m1FqHyVLfJjof2EFwBW4xOauNXHbGYnyEnisMkt3cKsNe2aNIw1lrg-CrgVrGW2IivyySWF5sov1HG0s/s320/EB1.jpg" /></a></div>
<p>
<center><b>
<p>
The binding site for End Binding protein 1 (highlighted in green) on the microtubule lattice at the corner of four tubulin dimers, visualised using cryo-electron microscopy
<br>
Image from Maurer <i>et al.</i> 2012, <i>Cell</i> <u>149(2)</u>:371-82. Full text <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368265/">here</a>.
</b></center>
<p>
Images of this structure have shown that the end binding proteins bind at the corner of four of the tubulin units. They have explained a lot of the properties of growing microtubules, but there is still more to learn. A full understanding will need structures that are at even higher resolution, where the positions of individual atoms can be made out. Following many years of technical development, today’s most powerful electron microscopes are now making this possible.
<p>
In the last section of the lecture, Moores left the topic of research to talk briefly about another of her passions: the promotion of women in science. She explained that although 65% of under-graduates in the biological sciences are now women, the proportion of women drops to 40% at any academic grade and 25% for full professors.
<p>
A study cited by the European Molecular Biology Organisation has suggested that the barriers for women scientists to progress are set so high that at the current rate of progress full equality would never be achieved. Birkbeck has signed up to the Athena SWAN Charter, set up to encourage higher education institutions to transform their culture and promote gender equality. She described her work with the Athena SWAN team that has so far resulted in the college gaining a bronze award as being as exciting as, but also as challenging as, her studies of microtubules.
<p>
<p>
<i>
There is more information about the structure and function of microtubules on <a href="http://pps14.cryst.bbk.ac.uk/course/section7/open2-cytoskeleton.html">this page</a> in section 7 of the PPS course. The technique of cryo-electron microscopy is covered in some detail in the second-year MSc module <a href="http://www.bbk.ac.uk/study/2015/postgraduate/programmes/TPCBIMOL_C/">Techniques in Structural Molecular Biology</a>.
</i>Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-49275293837833135832015-04-28T06:22:00.000-07:002015-04-28T06:28:37.809-07:00Protein Machines in the Molecular Arms RaceBirkbeck’s Science Week 2015 was held from Monday 23 to Thursday 26 March and included three evenings of public talks by senior researchers. The first two lectures, on the Tuesday, were given by two of the college’s most distinguished women scientists: <a href="http://www.bbk.ac.uk/biology/our-staff/academic/helen-saibil">Helen Saibil</a> from the Department of Biological Sciences and Karen Hudson-Edwards from Earth and Planetary Sciences; they were billed together as a ‘Women in Science Evening’.
<p>
The lectures were all introduced by the Dean of the Faculty of Science, <a href="http://www.bbk.ac.uk/biology/our-staff/academic/professor-nicholas-keep">Nicholas Keep</a> who described Saibil, a close colleague, as “our most eminent female scientist”. She came to Birkbeck from Canada via a PhD at King’s College London under the supervision of Nobel laureate <a href="http://www.nobelprize.org/nobel_prizes/medicine/laureates/1962/wilkins-facts.html">Maurice Wilkins</a> and post-doctoral work at Oxford.
<p>
Since arriving here in the 1980s she has built up an internationally renowned structural biology lab, focusing in particular on the technique of electron microscopy. She has been a Fellow of the <a href="https://royalsociety.org/about-us/fellowship/">Royal Society</a> since 2006 and of the <a href="http://www.acmedsci.ac.uk/fellows/">Academy of Medical Sciences</a> since 2009.
<p>
Saibil began her lecture by explaining that proteins can act as little machines, performing mechanical tasks that are essential for the maintenance of life. Her group has been interested for some time in proteins that can punch holes in the walls of cells. This allows the cell contents to leak out in a damaging process known as lysis, and it also allows toxins to enter the cells. These proteins can therefore be thought of as powerful weapons, and they are deployed on both sides of a ‘molecular arms race’: by pathogens and by the immune systems of humans and other animals.
<p>
Most soluble proteins fold into a single stable structure that tries, as far as possible, to keep their hydrophobic (“water-hating”) parts – the side chains of certain amino acids – in the interior of the protein, with the hydrophilic (“water-loving”) side chains on the outside, in contact with the watery environment inside or outside cells.
<p>
Pore-forming proteins, however, have a ‘Jekyll and Hyde’ like identity: they can form two distinctly different shapes, one as individual, soluble molecules and the other when they associate with each other into membrane-bound rings to form cylindrical pores. These structures, and the conformational change between them, are remarkably similar in proteins from bacteria and from the immune system.
<p>
Pore-forming toxins have been found in types of bacteria that are responsible for some deadly human diseases, including meningitis and pneumonia. The structure of a <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=1PFO">monomeric form</a> of one of these proteins in solution was first solved in 1998, using X-ray crystallography. However, large complexes of many protein molecules are more readily solved by electron microscopy, particularly when those complexes are embedded in membranes.
<p>
In 2005 Saibil and her group described structures of the pore-forming toxin <a href="http://en.wikipedia.org/wiki/Pneumolysin">pneumolysin</a> from <i>Streptococcus pneumoniae</i>, in complex with a model cell membrane. They found that the proteins formed two distinctly different ring-shaped structures. Initially, they formed into a ring sitting on top of the membrane, which was termed the <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=2BK2">pre-pore</a>; then they changed shape to burrow part of each protein deep into the membrane and form the <a href="http://www.rcsb.org/pdb/explore.do?structureId=2BK1">pore itself</a>. Each monomer in the pre-pore had a structure that was similar to that of the molecule in solution, but they underwent large structural changes to form the pore.
<P>
<center>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjrbgL8Te1sw8A-Njqb4kQHyKak5KAGj_gxakWI2WsSmljJzx2mhTSoMm0dizai65-JbsWFY6FTcEaz6Zo07ruqbOOoJds7mrLy5ZOVCM_ZVCsDREmF2HB1VTUtvgfztXMSSkwnd-nPjVo/s1600/HSsuilysingraphic_450w.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjrbgL8Te1sw8A-Njqb4kQHyKak5KAGj_gxakWI2WsSmljJzx2mhTSoMm0dizai65-JbsWFY6FTcEaz6Zo07ruqbOOoJds7mrLy5ZOVCM_ZVCsDREmF2HB1VTUtvgfztXMSSkwnd-nPjVo/s320/HSsuilysingraphic_450w.jpg" /></a></div>
<br>
<i>Schematic illustration of how suilysin, a bacterial cholesterol-dependent cytolysin, drills holes in cell membranes. Image © Adrian Hodel, London Centre for Nanotechnology</i>
</center>
<p>
Most structures solved by electron microscopy are at lower resolution than those solved by X-ray crystallography, and it is not possible to trace the positions of individual atoms at lower resolutions (e.g. worse than 3 A). Saibil and her colleagues were able to interpret the structure of the proteins making up the pore by fitting pieces of the X-ray structure of the isolated molecule into their electron density.
<p>
They found a dramatic change in structure, with the tall, thin protein structure collapsing into an arch and a helical region stretching out to form a long, extended beta hairpin. It is these hairpins that join together to form the walls of the pore. The process of pore forming therefore has three stages: firstly the toxin molecules bind to the surface of their target cells, then they associate into the circular pre-pores and finally they change shape in a concerted manner, punching holes in the cell membranes by ejecting a disc of membrane, letting other toxins in and cell contents out.
<p>
Saibil then turned the focus of her talk from attack by bacteria to the human immune system’s defence. <a href="http://en.wikipedia.org/wiki/Natural_killer_cell">Natural killer (NK) cells</a> are specialised lymphocytes (white blood cells) that kill virally-infected and cancerous cells in the bloodstream. They kill on contact with their target cells by releasing a toxic protein into those cells that stimulates those target cells to commit suicide in a process known as programmed cell death or apoptosis. We have only recently learned that the mechanism through which the NK cells work is very similar to the mechanism of the bacterial pore-forming toxins.
<p>
Natural killer cells express a protein called perforin that has a similar structure in solution to the bacterial pneumolysin. Although there is very little sequence similarity between these proteins – there is only one amino acid conserved throughout all the known bacterial and vertebrate proteins of this family, a glycine at a critical position for the conformational change – the structures are similar enough to suggest that the proteins all once had a common ancestor.
<p>
Saibil and her colleagues used electron microscopy to discover that this protein forms a pore through a similar mechanism to pneumolysin: the helical region that unfolds into the beta hairpin to form the pore forms the core of the molecular machine and is largely unchanged between the structures. There are some differences between the structures, however; in particular, there is no need for the perforin structure to ‘collapse’ as the molecule has ‘arms’ that are long enough to form the hairpin and punch the hole without bending into an arch.
<P>
The mechanism through which the NK cells kill their target cells is now quite well understood. When the two cells come into contact they form a temporary structure called an immune synapse that allows the pore to form and proteases called granzymes, which induce apoptosis, to enter the target cells. <a href="https://www.youtube.com/watch?v=MS9rRAO-SPo">This YouTube video</a> illustrates the natural killer cells’ mechanism of action, and <a href="https://www.youtube.com/watch?v=gKFXTJ9MjFg">this one</a> shows a detailed view of the immune synapse. Other, similar proteins have been identified in oyster mushrooms; these form more rigid structures that are easier to work with. Saibil’s group and their collaborators have been able to solve the structure of this protein in <a href="http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002049">intermediate stages of pore formation</a> and are beginning to gain an understanding of exactly how it unfolds.
<p>
<center>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj-b6zsLFnSk6lhPKI0MAHQHIgJ7nixvqHxT1o42bRYyg9fnjGZL1iabnmGh7MnKZpAnwkKze6Gxy-CmA_mnXVc-1cT-MSHCy2p8CtH3YOX1pGpZ8MIh-N-r6Y-ghG-8MqK0QuBLM0mQac/s1600/plyporemapfitilt.jpeg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj-b6zsLFnSk6lhPKI0MAHQHIgJ7nixvqHxT1o42bRYyg9fnjGZL1iabnmGh7MnKZpAnwkKze6Gxy-CmA_mnXVc-1cT-MSHCy2p8CtH3YOX1pGpZ8MIh-N-r6Y-ghG-8MqK0QuBLM0mQac/s320/plyporemapfitilt.jpeg" /></a></div>
<br>
<i>The pore of the oyster mushroom protein pleurotolysin, a member of the pneumolysin family. Image © Natalya Lukoyanova and Helen Saibil, from Lukoyanova <u>et al.</u>, <u>PLoS Biology</u> <b>13</b>:e1002049 </i>
</center>
<p>
Mutations in perforin that prevent it from functioning cause a rare disease called <a href="http://en.wikipedia.org/wiki/Hemophagocytic_lymphohistiocytosis">haemophagocytic lymphohistiocytosis</a>, which is almost invariably fatal in childhood. Understanding the mechanism of action of this important family of protein ‘weapons’ in both attack and defence may help find a cure for this devastating condition, as well as for some commoner disorders of the immune system and important infectious diseases.
<p>
<p>
<i>This post is cross-posted from the Birkbeck Events blog.
<p>
PPS students can learn much more about electron microscopy by taking the second-year module Techniques in Structural Molecular Biology to complete the MSc.</i> Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-75444812600308396252015-03-25T07:55:00.000-07:002015-03-25T07:55:23.873-07:00Claudins, allowing flow in a tight situation<h1>
<span lang="EN-US"><a href="http://www.sciencedirect.com/science/article/pii/S0022283614005713"><i><span style="font-size: 11.0pt; font-weight: normal; mso-ascii-font-family: Calibri; mso-ascii-theme-font: minor-latin; mso-bidi-font-weight: bold; mso-hansi-font-family: Calibri; mso-hansi-theme-font: minor-latin;">Suzuki,
H., Tani, K., Tamura, A., Tsukita, S. (2015) Model for the Architecture of
Claudin-Based Paracellular Ion Channels through Tight Junctions. J Mol Biol.
2015 Jan 30; 427(2):291-297</span></i></a></span><i><span lang="EN-US" style="color: #1f497d; font-size: 11.0pt; font-weight: normal; mso-ascii-font-family: Calibri; mso-ascii-theme-font: minor-latin; mso-bidi-font-weight: bold; mso-hansi-font-family: Calibri; mso-hansi-theme-font: minor-latin; mso-themecolor: text2;"><o:p></o:p></span></i></h1>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span lang="EN-US">Epithelial cells form a border between the
inside and outside of the body. They
cover both the external surface of the body (skin) and the lining of internal
cavities such as the lungs or the gastrointestinal tract. These cells are sealed together by <a href="http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/J/Junctions.html">tight
junctions</a>, which are just beneath the external surface.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">The tight junctions form a cell to cell
adhesion network that simultaneously blocks the passage of some substances
across the epithelia and allows the highly regulated movement of specific ions
and solutes across the barrier and between cells.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">The primary components of tight junctions
are claudins and recently there have been great advances in explaining their
structure and function. </span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">The first crystal structure of a single
claudin monomer, solved in 2014, is shown below. </span></div>
<div class="MsoNormal">
<span lang="EN-US">. </span><v:shapetype coordsize="21600,21600" filled="f" id="_x0000_t75" o:preferrelative="t" o:spt="75" path="m@4@5l@4@11@9@11@9@5xe" stroked="f">
<v:stroke joinstyle="miter">
<v:formulas>
<v:f eqn="if lineDrawn pixelLineWidth 0">
<v:f eqn="sum @0 1 0">
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgF3mFjbH-s5jr4UgUsmmZ-vEzqdws3xsbgDvDqjSlS4FPLs8buXydE6LucN0ZrZyMvayIZ5nQKZnDVW19aoMd9biIjeob42edDuYcnRgVcez0gvL0X0Jp9IEhU7ZXr2ivoXGrPuZYJDwEL/s1600/Claudins1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgF3mFjbH-s5jr4UgUsmmZ-vEzqdws3xsbgDvDqjSlS4FPLs8buXydE6LucN0ZrZyMvayIZ5nQKZnDVW19aoMd9biIjeob42edDuYcnRgVcez0gvL0X0Jp9IEhU7ZXr2ivoXGrPuZYJDwEL/s1600/Claudins1.jpg" /></a></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<i><span lang="EN-US">Image adapted from
</span><span lang="EN-US">(Suzuki, H. <u>et al</u>., (2014)).</span></i><span lang="EN-US"> </span><i><span lang="EN-US" style="background: #EAF2F8; color: #333333; mso-bidi-font-family: Helvetica; mso-bidi-font-size: 11.0pt;">Crystal structure of mouse
claudin-15 </span></i><span lang="EN-US"> (<a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4P79">PDB 4P79</a>)</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">The claudin <a href="http://en.wikipedia.org/wiki/Protomer">protomer</a> (monomer) has two
distinct sections: a tight bundle of
four α helices, which are embedded in the membrane of an epithelial cell, and
five β strands, which form a curved sheet on the extracellular side. These extracellular β sheets contain several
negatively charged residues which have previously been shown to be necessary
for both the formation of a tight barrier between cells and its selectivity for
positive ions (cations). They are also
thought to form the paracellular channels that traverse the epithelium. </span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">Two of these functions have been
demonstrated by elegant experiments that substituted the acidic residues on the
β sheet with positively charged ones, resulting in paracellular channels that
were selective for anions.</span><i><span lang="EN-US"><o:p></o:p></span></i></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">The monomer structure alone, however, could
not provide explanations for either the structure of tight junction strands,
which form the mesh that creates cell adhesion, or the transcellular channels
that allow interaction between cells.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">A recent study by the same team that solved
the first claudin structure, <a href="http://www.sciencedirect.com/science/article/pii/S0022283614005713">(Suzuki,
H. et al., (2015))</a>, uses cysteine crosslinking experiments to suggest an
arrangement of the claudin monomers which would produce paracellular channels
which run across the epithelium, transcellular channels which are perpendicular
to the paracellular channels, and linear tight junction strands. </span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">The proposed model uses a basic building
block of two claudin monomers lying alongside each other but antiparallel so
that the outer edges of the β sheets connect via hydrogen bonds to form half of
a β barrel structure. These dimers then
associate to form long polymers of the double row of claudins (see figure).</span></div>
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<span lang="EN-US"><br /></span></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjsld4g-EyN-unx92bgFfmhB9VXWm29J6T6HGOvCIzi_xQaMQZyLC485cL3z8e9p-1X1XDrkVd1QtTP60tTqoJvl07GgZ32NZtCzn3ndQYIax10xPMKwzrpzzmN_S0hLm0dJAOVJSFDYT4j/s1600/Claudins2.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjsld4g-EyN-unx92bgFfmhB9VXWm29J6T6HGOvCIzi_xQaMQZyLC485cL3z8e9p-1X1XDrkVd1QtTP60tTqoJvl07GgZ32NZtCzn3ndQYIax10xPMKwzrpzzmN_S0hLm0dJAOVJSFDYT4j/s1600/Claudins2.jpg" height="320" width="216" /></a></div>
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<span lang="EN-US"><br /></span></div>
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<i><span lang="EN-US">Image adapted
from </span><span lang="EN-US">(Suzuki, H. <u>et al</u>., (2015)).</span></i><span lang="EN-US"> <i>The first image shows the association of claudin monomers and their
polymerization. The second image shows
the same structure rotated through 90˚ so that we are looking down the half β
barrel .<o:p></o:p></i></span></div>
<div class="MsoNormal">
<span lang="EN-US"><i><br /></i></span></div>
<div class="MsoNormal">
<span lang="EN-US">This model was tested by substituting Cys
residues for key Asn positions at the proposed interface between the outer β
strands. The mutant claudins were found
to form dimers that reverted to ther usual monomeric structure in reducing
conditions. This illustrates that the
molecules normally associate face to face, along the proposed β strands, so that once the substitutions were
made disulphide bridges could be formed.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">More corroboration was obtained using
electron microscopy (EM), which is studied in the second-year <a href="http://www.bbk.ac.uk/study/2015/postgraduate/programmes/TPCBIMOL_C/">TSMB
course</a>. A form of EM known as <a href="http://en.wikipedia.org/wiki/Electron_microscope">freeze fracture
electron microscopy</a>, which is often used to examine proteins embedded in
lipid membranes, was used to provide images of tight junction strands, which appear
as a network of linear strands whose width is consistent with the model.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">In the epithelial sheet, a claudin double
row would be anchored in the cell membrane with its half β barrel projecting
out. By lining itself up against a
similarly projecting double row in the adjacent cell we can see how tight
junction strands with paracellular channels could be formed. The diameter of this channel would be less
than 10 Å, enough to allow the passage of hydrated ions but to restrict their
flow.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjUdgXhUIQCT71HKpTYRm8iVIFwpQs9S9zCoW7XlPjavERLduhJUl0YFJTlApzal3a1D4eVAG5ngVJuEdPdbXYf0X0P5n0Qcg_w3B2YNymEa4NXHsrXJ0e1lkMvCQZgTo5AscEh4bBCP9fN/s1600/Claudins3.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjUdgXhUIQCT71HKpTYRm8iVIFwpQs9S9zCoW7XlPjavERLduhJUl0YFJTlApzal3a1D4eVAG5ngVJuEdPdbXYf0X0P5n0Qcg_w3B2YNymEa4NXHsrXJ0e1lkMvCQZgTo5AscEh4bBCP9fN/s1600/Claudins3.jpg" /></a></div>
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<span lang="EN-US"><br /></span></div>
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<span lang="EN-US"><br /></span></div>
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<div class="MsoNormal">
<i><span lang="EN-US">Image adapted
from </span><span lang="EN-US">(Suzuki, H. <u>et al</u>., (2015)).</span></i><span lang="EN-US"> <i>The image shows the joining of a row of half β barrels from adjacent
cells, forming a row of paracellular channels.<o:p></o:p></i></span></div>
<div class="MsoNormal">
<span lang="EN-US"><i><br /></i></span></div>
<div class="MsoNormal">
<span lang="EN-US">Epithelial cells maintain the separation of
several internal spaces within the body and each of the interfaces has its own
requirements for substances that are allowed to cross the border. This means that the paracellular channels
need different ion specificity depending on which boundary they maintain. This could be provided by the charges and
orientation of the residues in the channel lining and may also be influenced by
flexible loops that link the β strands.
It is also possible that these loops, which are not well conserved among
the 27 members of the claudin family in the mouse (or human) proteome, could
function as recognition regions so that only claudins of the same type can
associate.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">Possibly the most remarkable aspect of this
model, however, can be seen by rotating it through 90˚.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiOw3U8d7daDadqpVkd5ZAj5x0S-uo9A9fQxb9Jck3pv_Y_6N1NB7H8kjkS8XOG-TzJ5CPUR87hXVSnXCbPE1MzuRipjXj0mmKM1CEOZ3n5VElILnrWwtjOyFMpGpOAZLSg47m1-IHM7SrJ/s1600/Claudins4.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiOw3U8d7daDadqpVkd5ZAj5x0S-uo9A9fQxb9Jck3pv_Y_6N1NB7H8kjkS8XOG-TzJ5CPUR87hXVSnXCbPE1MzuRipjXj0mmKM1CEOZ3n5VElILnrWwtjOyFMpGpOAZLSg47m1-IHM7SrJ/s1600/Claudins4.jpg" /></a></div>
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<span lang="EN-US"><br /></span></div>
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<div class="MsoNormal">
<i><span lang="EN-US">Image adapted
from </span><span lang="EN-US">(Suzuki, H. <u>et al</u>., (2015)).</span></i><span lang="EN-US"> <i> In this image the paracellular channels are
seen in magenta whilst the α helices are aligned perpendicular to the
page. <o:p></o:p></i></span></div>
<div class="MsoNormal">
<span lang="EN-US"><i><br /></i></span></div>
<div class="MsoNormal">
<span lang="EN-US">Further gaps can now be seen; these could
provide transcellular channels allowing the flow of ions and solutes between
epithelial cells.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<div class="MsoNormal">
<span lang="EN-US">In short this model appears to provide
solutions to all of the functions required by claudins in tight junctions
between epithelial cells and it will be very interesting to see if it proves to
be correct as further crystal structures
are solved.</span></div>
<div class="MsoNormal">
<span lang="EN-US"><br /></span></div>
<br />
<div class="MsoNormal">
<span lang="EN-US">Do look at the original paper if you have
the chance as it includes an excellent short animation that shows the
association of single claudin monomers into tight junction strands and then rotates
the structure to show the different channels. (Follow the web link to the
Supplementary Data.) </span></div>
Jill Fairclothhttp://www.blogger.com/profile/09598549871290029938noreply@blogger.com1tag:blogger.com,1999:blog-7005104906174355459.post-76422727914716784822015-01-07T08:44:00.002-08:002015-01-07T08:44:50.351-08:00From Crystallography to Light (according to the United Nations)Those of you who started the PPS course in 2014 may not have realised that they began their studies of structural biology at an auspicious time for this subject. Last year was designated the International Year of Crystallography to mark the centenaries of both the discovery of X-ray diffraction by crystals (first published in 2013) and the award, in 2015, of the <a href="http://www.nobelprize.org/nobel_prizes/physics/laureates/1915/">Nobel Prize</a> to the father-and-son team of William and Lawrence Bragg who made the discovery. I have blogged about this in much more detail over the previous year, with a post in <a href="http://principlesofproteinstructure.blogspot.co.uk/2013/12/a-very-short-history-of-crystallography.html">December 2013</a> on "a very short history of crystallography" and one in <a href="http://principlesofproteinstructure.blogspot.co.uk/2014/07/science-week-2014-birkbeck-and-history.html">July</a> reporting on lectures by two of our distinguished emeritus professors, Paul Barnes and David Moss. Professor Moss took the subject up to date with a race through the history of structural biology, from the publication of the first protein structures - myoglobin and haemoglobin - in 1958 to the 100,000th structure that entered the <a href="http://rcsb.org/pdb/home/home.do">Protein Data Bank</a> in 2014. If you want to read more about the numerous events and publications around the world that honoured and publicised crystallographers during 2014, the best place to start is probably the official website of <a href="http://www.iycr2014.org/">IYCr2014</a>. And if you have access to <i><a href="http://www.rsc.org/chemistryworld/">Chemistry World</a></i>, the membership magazine of the Royal Society of Chemistry, you can read two more pieces by me: "Crystal Clear" in the January 2014 issue and "Life in the Freeze Frame", featuring structural biology, in September. (Unfortunately, it is not available in the Birkbeck e-library.)
<p>
2014 was designated as the International Year of Crystallography by the United Nations, as part of its programme of using anniversaries to highlight topics and issues that it sees as important. There have been many international years on science-based topics, some broader than others: 2005, for example, was the International Year of Physics. And in this new year the focus moves on to another physical science topic. 2015 is the <a href="http://www.light2015.org/Home.html">International Year of Light</a>: or more precisely that "... of Light and Light-based Technologies".
<p>
And optics, the science of light, is very closely related to crystallography. Both light and X-rays are parts of the <a href="https://en.wikipedia.org/wiki/Electromagnetic_spectrum">electro-magnetic spectrum</a> and as such are both particles and waves; the differences in their frequencies and wavelengths lead to their very different properties. The first X-ray diffraction experiments were conducted mainly to find out whether the hypothesis that X-rays were electromagnetic waves with a wavelength of about 1 Angstrom was correct. The utility of this technique as a probe for molecular structure was initially a side effect of the similarity between this wavelength and the length of a covalent bond. (So-called "copper K-alpha" radiation, which is often used in crystallography, has a wavelength of 1.54Å, which is exactly equivalent to the length of a single C-C bond.) And the basic equation that defines X-ray diffraction - Bragg's Law, or nλ = 2d sin θ - applies to all electromagnetic radiation, not just to X-rays.
<p>
Advances in optical technology have been responsible for some important advances in crystallography. The discovery, in the mid- twentieth century, that charged particles emit electromagnetic radiation in the form of X-rays when accelerated radially was initially viewed as a problem of energy loss. This "lost" radiation, which is orders of magnitude more intense than the X-rays used in the first diffractometers, was soon exploited in synchrotron radiation sources and most non-routine protein structures are now solved using synchrotron radiation. The UK's synchrotron source, a doughnut-shaped structure half a kilometre across located at Harwell in Oxfordshire, is even known officially as "<a href="http://www.diamond.ac.uk/Home.html">Diamond Light Source</a>". And recent advances in optics are gradually reducing the size of intense X-ray sources so that it will become possible to do complex structural biology that now needs a synchrotron using a lab-based machine.
<p>
The next big technical advance to influence X-ray crystallography is the <a href="https://en.wikipedia.org/wiki/Free-electron_laser">free electron laser</a>, which can generate pulses of X-rays that are much more intense than those produced by even the largest synchrotrons. This is a beam of electrons that is accelerated to almost the speed of light through a side-to-side magnetic field to produce pulses of extremely intense electromagnetic radiation. These pulses are exceptionally short, each lasting only a few femtoseconds (1 fs = 10<sup>-15</sup>s). This is a feature of the technology that leads to two advantages: the crystals remain intact, even though they are exposed to extremely intense radiation, and it becomes possible to take "snapshot" structures very close together in time study the dynamic behaviour of molecules. Free electron laser radiation also enables precise structures to be obtained from much smaller crystals, which makes large complexes and membrane-bound proteins more tractable to crystallography. And it may one day be possible, using this technique, to determine a structure from the smallest "crystal" of all - a single molecule - and thus liberate protein crystallography from one of its most important bottlenecks: the need to grow protein crystals.
<p>
<p>
<i><u>Acknowledgement:</u>This post owes much to <a href="https://crystallography365.wordpress.com/2015/01/01/from-crystallography-to-light/">this one</a> on the "Crystallography 365" blog (author unknown)</i>Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-30867034831067899032014-10-13T09:24:00.000-07:002014-10-13T09:24:28.919-07:00Welcome to PPS students 2014-15!This post is extremely like those I have written at this time of year for the past few years. This is because what I have to say now is very, very similar...
<p>
I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have just started studying Birkbeck's Principles of Protein Structure course, and a welcome back to any who have taken a break in studies and intend to
complete the course this year.
<p>
I run this blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. Throughout the taught course (but more often in the later part of the course) I will post reports of recent developments. I might, example, report on talks given in the ISMB seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The overall title of the programme for Autumn 2014 is <i><a href="http://www.ismb.lon.ac.uk/seminar.html">Synthetic Biology</a></i>: an innovative and important topic that relates quite closely to some of the material we cover in the later sections of the course, particularly the sections on Bioinformatics and the Protein Lifecycle. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas.
<p>
Some of the posts on this blog are written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in <a href="http://principlesofproteinstructure.blogspot.co.uk/2012_03_01_archive.html">this post</a> written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.
<p>
Do, if you get a chance, look through some of the earlier blog posts to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!
<p>
I particularly recommend that you look at a couple of posts from last academic year - <a href="http://principlesofproteinstructure.blogspot.co.uk/2013/12/a-very-short-history-of-crystallography.html">December 2013</a> and <a href="http://principlesofproteinstructure.blogspot.co.uk/2014/07/science-week-2014-birkbeck-and-history.html">July 2014</a> - about the history of structural science, particularly X-ray crystallography. Crystallography was the first method to be developed for solving the structure of biological macromolecules, and it is still the most important. The year 2014 was designated by the United Nations as the <a href="http://www.iycr2014.org/">International Year of Crystallography</a>, marking the year between the centenaries of the publication of the first papers on X-ray diffraction and the award of the <a href="http://www.nobelprize.org/nobel_prizes/physics/laureates/1915/">1915 Nobel Prize for Physic</a>s to the father-and-son team of William and Lawrence Bragg who made the principal discoveries. The International Year has been marked by a wide range of activities, special symposia, publications, "open labs" and even postage stamps, and there are still a few events planned.
<p>
So - the best of luck for the 2014-15 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our MSc in Structural Molecular Biology.
<p>
Best wishes,
<p>
Dr Clare Sansom
Senior Associate Lecturer, Biological Sciences, Birkbeck and Tutor, Principles of Protein Structure Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com0tag:blogger.com,1999:blog-7005104906174355459.post-41461598767716955512014-07-24T06:01:00.001-07:002014-07-28T08:26:13.471-07:00Science Week 2014: Birkbeck and the History of CrystallographyScience Week at Birkbeck in 2014 featured two lectures on Department of Biological Sciences, both presented on 2 July. One of these was a double act from two distinguished emeritus professors and Fellows of the College, <a href="http://img.chem.ucl.ac.uk/www/barnes/homepage.htm">Paul Barnes</a> and <a href="http://people.cryst.bbk.ac.uk/~ubcg05m/">David Moss</a>. Remarkably, they both started their working lives at Birkbeck on the same day – 1 October 1968 – and so had clocked up over 90 years of service to the college between them by Science Week 2014.
<p>
The topic they took was a timely one: the history of the science of crystallography over the past 100 years. UNESCO has declared 2014 to be the <a href="http://www.iycr2014.org/">International Year of Crystallography</a> in recognition of the seminal discoveries that started the discipline, which were made almost exactly 100 years ago; a number of the most important discoveries of that century were made by scientists with links to Birkbeck.
<p>
The presenters divided the “century of crystallography” into two, with Barnes speaking first and covering the first 50 years. In giving his talk the title “A History of Modern Crystallography”, however, he recognised that crystals have been observed, admired and studied for many centuries. What changed at the beginning of the last century was the discovery of X-ray diffraction. <a href="http://www.nobelprize.org/nobel_prizes/physics/laureates/1901/">Wilhelm Röntgen</a> was awarded the first-ever Nobel Prize for Physics for his discovery of X-rays in 1896, but it was almost two decades before anyone thought of directing them at crystals. The breakthroughs came when <a href="http://www.nobelprize.org/nobel_prizes/physics/laureates/1914/">Max von Laue</a> showed that a beam of X-rays can be diffracted by a crystal to yield a pattern of spots, and the father-and-son team of <a href="http://www.nobelprize.org/nobel_prizes/physics/laureates/1915/">William Henry and William Lawrence Bragg</a> showed that it was possible to derive information about the atomic structure of crystals from their diffraction patterns. These discoveries also solved – to some extent – the debate about whether X-rays were particles or waves, as only waves diffract; we now know that all electromagnetic radiation, including X-rays, can be thought of as both particles and waves
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Von Laue and the Braggs were awarded Nobel Prizes for Physics in 1914 and 1915 respectively, and between 1916 and 1964 no fewer than <a href="http://www.iucr.org/news/newsletter/volume-16/number-3/crystallography-and-the-nobel-prize">13 more Nobel Prizes</a> were awarded to 18 more scientists for discoveries related to crystallography. <a href="http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1936/">Petrus Debye</a>, who won the Chemistry prize in 1936, showed how to quantify the thermal motion of atoms as vibrations within a crystal. He also invented one of the first powder diffraction cameras, used to obtain diffraction patterns from powders of tiny crystallites. Another Nobel Laureate, <a href="http://www.nobelprize.org/nobel_prizes/physics/laureates/1946/">Percy Bridgman</a>, studied the structures of materials under pressure: it has been said that he would “squeeze anything he could lay his hands on”, often up to intense pressures.
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Scientists and scientific commentators often argue about which of their colleagues would have most deserved to win the ultimate accolade. Barnes named three who, he said, could easily have been Nobel Laureates in the field of crystallography. One, <a href="http://en.wikipedia.org/wiki/Paul_Peter_Ewald">Paul Ewald</a>, was a theoretical physicist who had studied for his PhD under von Laue in Munich, and the other two had strong links with Birkbeck. <a href="http://en.wikipedia.org/wiki/John_Desmond_Bernal">JD “Sage” Bernal</a> was Professor of Physics and then of Crystallography here; he was famous for obtaining, with <a href="http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1964/">Dorothy Crowfoot (later Hodgkin)</a> the first diffraction pattern from a protein crystal, but his insights into the atomic basis of the very different properties of carbon as diamond and as graphite were perhaps even more remarkable. He took on <a href="http://en.wikipedia.org/wiki/Rosalind_franklin">Rosalind Franklin</a>, whose diffraction patterns of DNA had led Watson and Crick to deduce its <a href="http://www.nobelprize.org/educational/medicine/dna_double_helix/readmore.html">double helical structure</a>, after she left King’s College, and she did pioneering work on virus structure here until her premature death in 1958.
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Barnes ended his talk and led into Moss’s second half-century with a discussion of similarities between the earliest crystallography and today, as now, you only need three things to obtain a diffraction pattern: a source of X-rays, a crystalline sample, and a recording device; the differences all lie in the power and precision of the equipment used. He demonstrated this with a “symbolic demo” that ended when he pulled a model structure of a zeolite out of a large cardboard box.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEisAgBofwjMADXsZryAh2J7fJD8eafRILXop0sP_ApW6aaH067p_G_OCLp_Z_-BENxfL8sV2bzhQZ6FwdJgQFGgzuECDOpB1B3ory6pR6SLveeteZsoQwVxnNMtFz1c4GzakdEITEQt1gE/s1600/barnes-demo.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEisAgBofwjMADXsZryAh2J7fJD8eafRILXop0sP_ApW6aaH067p_G_OCLp_Z_-BENxfL8sV2bzhQZ6FwdJgQFGgzuECDOpB1B3ory6pR6SLveeteZsoQwVxnNMtFz1c4GzakdEITEQt1gE/s320/barnes-demo.jpg" /></a></div>
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<center><i>Paul Barnes demonstrates the basic principles of X-ray crystallography using a large cardboard box. Photo © Harish Patel and Ruben Zamora, Department of Psychological Sciences, Birkbeck</i></center>
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David Moss then took over to describe some of the most important crystallographic discoveries from the last half-century. His talk concentrated on the structures of large biological molecules, particularly proteins, and he began by explaining the importance of protein structure. All the chemistry that is necessary for life is controlled by proteins, and knowing the structure of proteins enables us to understand, and potentially also to modify, how they work.
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Even the smallest proteins contain thousands of atoms; in order to determine the position of all the atoms in a protein using crystallography you need to make an enormous number of measurements of the positions and intensities of X-ray spots. The process of solving the structure of a protein is no different from that of solving a small molecule crystal structure, but it is more complex and takes much more time. Very briefly, it involves crystallising the protein; shining an intense beam of X-rays on the resulting crystals to produce diffraction patterns, and then doing some extremely complex calculations. The first protein structures, obtained without the benefit of automation and modern computers, took many years and sometimes even decades.
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Thanks to Bernal’s genius, energy and pioneering spirit, Birkbeck was one of the first institutes in the UK to have all the equipment that was needed for crystallography. This included some of the country’s first “large” computers. One of the first electronic stored-program computers was developed in <a href="http://www.dcs.bbk.ac.uk/about/history/booth.php">Donald Booth’s laboratory</a> here in the 1950s. In the mid-1960s the college had an ATLAS computer with a total memory of 96 kB. It occupied the basements of two houses in Gordon Square, and crystallographers used it to calculate electron density maps of small molecules. Protein crystallography only “took off” in the 1970s with further improvements in computing and automation of much of the experimental technique.
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Today, protein crystallography can almost be said to be routine. The first step, crystallising the protein, can still be an important bottleneck, but data collection at powerful synchrotron X-ray sources is extremely rapid and structures can be solved quite easily with user-friendly software that runs on ordinary laptops. There are now over 100,000 protein structures freely available in the <a href="http://www.rcsb.org/pdb/home/home.do">Protein Data Bank (PDB)</a>, and about 90% of these were obtained using X-ray crystallography. The techniques used to obtain the other 10,000 or so, nuclear magnetic resonance and electron microscopy, are more specialised.
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Moss ended his talk by describing one of the proteins solved in his group during his long career at Birkbeck: a bacterial toxin that is responsible for the disease <a href="http://en.wikipedia.org/wiki/Gas_gangrene">gas gangrene</a> (PDB <a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=1CA1">1CA1</a>). This destroys muscle cells by punching holes in their membranes, and its victims usually have to have limbs amputated to save their lives. Knowing the structure has allowed scientists to understand how this toxin works, which is the first step towards developing drugs to stop it. But you can learn even more about how proteins work if you also understand how they move. Observing and modelling protein motion in “real time” still poses many challenges for scientists as the second century of crystallography begins.
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<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhlPWYxiYgTsCYu5UKo0bacswRBAWr2cslygMH-lnh0LJ0jYKiRUQHyRpbAdJwSydWsnmigw9Dr8MxDdEhXJ7a2-ztH6lKt11NYdisSfO8ePnnHU45yNTGYyznM_8AYgdqXkEueVcKOBQg/s1600/1CA1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhlPWYxiYgTsCYu5UKo0bacswRBAWr2cslygMH-lnh0LJ0jYKiRUQHyRpbAdJwSydWsnmigw9Dr8MxDdEhXJ7a2-ztH6lKt11NYdisSfO8ePnnHU45yNTGYyznM_8AYgdqXkEueVcKOBQg/s320/1CA1.jpg" /></a></div>
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<center>
<i>Structure of alpha-toxin, the key <u>Clostridium perfringen</u> toxin in gas gangrene. Image from the PDB.</i>
</center>Dr Clare Sansomhttp://www.blogger.com/profile/11905698604241444028noreply@blogger.com1