Crystallography: from Chocolate to Drug Discovery

Birkbeck has already established lecture series in honour of some of its most distinguished alumni. Until 2016, however, Rosalind Franklin – 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 Athena SWAN programme and will always be given by a distinguished woman scientist. And fittingly, the inaugural lecture, which was part of Science Week 2016, was devoted to Rosalind Franklin’s own discipline, crystallography. Elspeth Garman, 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.

Garman began her lecture by showing a short video 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 over 30’ long or too small to be visible with the naked eye.

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 Nobel Prize for Physics in 1915, 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.

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.

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.

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. Dorothy Hodgkin, 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 ribosome, 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.

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 Aaron Klug cited her inspiration in his own Nobel lecture in 1982. 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 Daily Express.

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 Diamond Light Source 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.

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 Protein Data Bank, 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.

Microtubules and Microscopes: Exploring the Cytoskeleton

Electron microscopist Carolyn Moores, the most recently appointed professor in the Department of Biological Sciences at Birkbeck, gave her inaugural lecture at the college on June 1.

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.

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.

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 John Kendrick-Jones’ lab at the world-famous Laboratory for Molecular Biology (LMB) in Cambridge. She then moved to work as a post-doc with Ron Milligan at the Scripps Research Institute in La Jolla, California, USA, and it was there that she began her studies on microtubules.

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 Helen Saibil, one of the UK’s principal exponents of that technique; she had known Nicholas Keep, 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 Institute for Structural Molecular Biology under the ‘inspired’ leadership of Gabriel Waksman.

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 microtubules. 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.

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.

Microtubules are built up from many copies of a small protein called tubulin, 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.

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 cell division 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.

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.

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.

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.

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
Image from Maurer et al. 2012, Cell 149(2):371-82. Full text here.

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.

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.

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.

There is more information about the structure and function of microtubules on this page in section 7 of the PPS course. The technique of cryo-electron microscopy is covered in some detail in the second-year MSc module Techniques in Structural Molecular Biology.