From 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 Nobel Prize 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 December 2013 on "a very short history of crystallography" and one in July 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 Protein Data Bank 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 IYCr2014. And if you have access to Chemistry World, 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.)

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 International Year of Light: or more precisely that "... of Light and Light-based Technologies".

And optics, the science of light, is very closely related to crystallography. Both light and X-rays are parts of the electro-magnetic spectrum 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.

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 "Diamond Light Source". 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.

The next big technical advance to influence X-ray crystallography is the free electron laser, 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^-15s). 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.

Acknowledgement:This post owes much to this one on the "Crystallography 365" blog (author unknown)

Science Week 2014: Birkbeck and the History of Crystallography

Science 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, Paul Barnes and David Moss. 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.

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 International Year of Crystallography 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.

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. Wilhelm Röntgen 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 Max von Laue 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 William Henry and William Lawrence Bragg 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

Von Laue and the Braggs were awarded Nobel Prizes for Physics in 1914 and 1915 respectively, and between 1916 and 1964 no fewer than 13 more Nobel Prizes were awarded to 18 more scientists for discoveries related to crystallography. Petrus Debye, 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, Percy Bridgman, 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.

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, Paul Ewald, was a theoretical physicist who had studied for his PhD under von Laue in Munich, and the other two had strong links with Birkbeck. JD “Sage” Bernal was Professor of Physics and then of Crystallography here; he was famous for obtaining, with Dorothy Crowfoot (later Hodgkin) 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 Rosalind Franklin, whose diffraction patterns of DNA had led Watson and Crick to deduce its double helical structure, after she left King’s College, and she did pioneering work on virus structure here until her premature death in 1958.

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.

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

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.

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.

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 Donald Booth’s laboratory 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.

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 Protein Data Bank (PDB), 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.

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 gas gangrene (PDB 1CA1). 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.

Structure of alpha-toxin, the key Clostridium perfringen toxin in gas gangrene. Image from the PDB.

From Genome to Proteome: BCA Winter Meeting 2012

The British Crystallographic Association is the main UK organisation supporting the science of crystallography in all its forms. Every year, its Biological Structures Group holds a meeting in the run-up to Christmas to discuss and celebrate recent developments in structural biology research. In 2012, this Winter Meeting was held at the MRC Laboratory of Molecular Biology at the University of Cambridge.

The LMB, as it is usually known, is one of the birthplaces of modern structural and molecular biology. It moved into its current building in 1962, the year when four of its most famous scientists were awarded two Nobel Prizes for some of the most important discoveries in twentieth-century biology: James Watson and Francis Crick or the structure of DNA, and Max Perutz and John Kendrew for the very first three-dimensional structures of proteins (myoglobin and haemoglobin, respectively).

It was appropriate, therefore, that the theme of this year's Winter Meeting was "From Genome to Proteome". The basic molecular processes that underlie all of life - DNA replication, transcription of DNA into RNA and translation of RNA into protein - are all, now, quite well understood. These processes are all very complicated and require numerous proteins, many of which interact together to form complexes and "molecular machines" that are quite large, at least in molecular terms. Scientists presenting at the meeting discussed recent, innovative studies of the structures of many of these proteins and the nucleic acids that they interact with. Many of these processes will be discussed in some detail in section 8 of the PPS course, "The Protein Lifecycle".

The meeting programme was divided into three sections, corresponding respectively to DNA synthesis and repair, RNA transcription and protein translation.

DNA Replication and Repair

DNA synthesis and repair are not even mentioned in the famous Central Dogma of Molecular Biology (put very simplistically, DNA makes RNA makes protein) but they are, of course, essential for it. The first speaker in this session, and therefore in the meeting as a whole, was Luca Pellegrini from the University of Cambridge. He described structural studies of the first part of this process: the initiation of DNA synthesis. In all organisms, this process involves an enzyme called primase, which is found at the DNA replication fork - the point at which the strands of the original DNA helix divide so that a new strand can be synthesised on each of the template strands. Pellegrini and his group have solved the structure of several of the subunits of yeast primase, alone and bound to part of the DNA polymerase Pol alpha, and are using these structures to deduce the precise mechanism of this vitally important process.

Then Neil Kad of the University of Essex described the techniques he has developed for visualising individual molecules, and how he is applying them to the study of DNA repair by nucleotide excision. Briefly, this technique involves stretching a single molecule of DNA between two positively charged silica beads, and tagging individual molecules of DNA-binding proteins using fluorescent quantum dots so that their binding to and progress along this DNA "tightrope" can be monitored. He has discovered that although single subunits of the Uvr DNA repair protein complex may bind DNA and search it for errors, a complex between the subunits UvrA and UvrB is required for quick and efficient searching.

Schematic diagram of a "DNA tightrope" with labelled proteins bound. (c) Neil Kad, from the Kad Lab homepage

Transcription

The spliceosome is a "molecular machine" comprised of protein and small nuclear RNA (snRNA) subunits that found only in eukaryotes and that catalyses the removal of introns from the messenger RNA precursor molecules that are initially transcribed from DNA. Chris Oubridge, a member of Kiyoshi Nagai's group at the MRC Laboratory of Molecular Biology in Cambridge (and therefore one of the "home team") described an atomic resolution structure of a complex known as U1 that forms a major part of the soliceosome. This "small nuclear ribonucleoprotein" (snRNP) comprises the snRNA molecule U1 bound to ten proteins. This technically challenging exercise in X-ray crystallography is yielding important insights into the function and mechanism of this important part of the spliceosome.

Structure of the U1 ribonucleoprotein, from Kiyoshi Nagai's web pages at the MRC-LMB.

Another interesting presentation in the Translation section was given by David Lilley from the University of Dundee, who described the structures of kink turns in RNA molecules, and how these structural motifs interact with proteins.

Translation

Since the modern Laboratory o Molecular Biology was constituted as the "Unit for Research on the Molecular Structure of Biological Systems'" in 1947, nine Nobel prizes have been awarded to scientists working there. Its most recent laureate, Venki Ramakrishnan, shared the 2009 chemistry prize with Tom Steitz from the US and Ada Yonath from Israel for determining the first atomic resolution structure of the ribosome. Israel Sanchez from Ramakrishnan's lab at the LMB gave a presentation on the mechanism by which stop codons, which give the signal to terminate protein synthesis, are decoded on the ribosome. This process, which occurs when one of the stop codons (UAA, UAG and UGA in the standard genetic code) binds to the ribosomal A site, is still less well understood than the process through which "sense" codons are decoded into amino acids. Sanchez and his colleagues are studying the structure and function of ribosomes bound to modified RNA in which the uridine in the first position of a stop codon has been substituted by pseudo-uridine. They have discovered that the decoding centre of the ribosome is more flexible than they had originally thought, an insight that may help the understanding of the termination of protein synthesis further.

The final speaker was Birkbeck's own Cara Vaughan. She discussed some of her recent research using a combination of X-ray crystallography and electron microscopy to decipher the assembly of the kinetochore. This is a structure that forms in eukaryotic cells during cell division and that links the dividing chromosome to the mitotic spindle. Vaughan's research concerns a protein called Hsp90 that activates many signalling proteins. This protein is a member of a class of proteins termed the chaperones, which are generically involved in the folding, unfolding and activation of other proteins. Vaughan and her co-workers have solved the structure of two interacting proteins found in yeast, Sgt1 and Skp1, which togethe3r seem to hold Hsp90 in an open conformation that enables other kinetochore proteins to bind.

Image of a dividing eukaryotic cell. The chromosomes are shown in blue, the microtubules of the mitotic spindle in green, and the kinetochores in pink. Image from Wikimedia Commons.

The annual Winter Meeting is the most high profile event organised by the Biological Structures Group of BCA. The association as a whole organises many other events, including, this year, the annual European Crystallographic Meeting. ECM 28 will be held at the University of Warwick from 25-29 August 2013; it will provide an opportunity for British and European crystallographers to celebrate the origin of their science with the discovery of X-ray diffraction by father and son William Henry and William Lawrence Bragg, almost exactly a hundred years ago.