Crystallography: From Chocolate to Drug Discovery

This post was contributed by Dr Clare Sansom, senior associate lecturer in the Department of Biological Sciences. Dr Sansom attended inaugural Rosalind Franklin Lecture during Birkbeck Science Week 2016.

Rosalind Franklin - slide

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.

Exploring crystals

Garman began her lecture by showing a short video that she had produced for 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.

Professor Nick Keep and Professor Elspeth Garman at the inaugural Rosalind Franklin lecture

Professor Nick Keep and Professor Elspeth Garman at the inaugural Rosalind Franklin lecture

Protein crystallography

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.

Real world applications

Dr Rosalind Franklin

Dr Rosalind Franklin

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.

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Crystallography: past, present and future (Science Week 2014)

This post was contributed by Dr Clare Sansom, Senior Associate Lecturer in Birkbeck’s Department of Biological Sciences

Prof Paul Barnes sets the scene for one of the experiments he carried out in the Crystallography lecture

The second of the Science Week lectures from the Department of Biological Sciences, which was presented on 2 July 2014, 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 Bragg 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. Then, 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.

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, 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. 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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