StrapX-ray crystallographyin here like this Complexity crystallised Protein x-ray crystallography has come a long way from a 12 year search for the structure of a single protein. Philip Ball reports

When John Kendrew walked into anticipated. It was not until 12 understanding how it does its often Max Perutz’s room at the Cavendish In short years later that Kendrew finally extremely precise and selective laboratory in Cambridge and said he  50 years ago, the worked out the structure of job: binding and transforming its wanted to be his research student, publication of the crystal myoglobin, the protein that stores substrate, for instance, or sticking Perutz was flattered but also structure of myoglobin oxygen temporarily in cells before to nucleic acids, inducing motion, flustered. For one thing, Kendrew – a protein extracted transferring it to haemoglobin, the or supplying a robust fabric in was almost Perutz’s age, and he from whale muscle – set oxygen-ferrying molecule in the tissues. And the rational design of looked rather grand in his Wing a landmark in structural bloodstream. Kendrew’s picture drugs that can bind to and modify Commander’s uniform. This was biology of myoglobin was detailed enough the behaviour of enzymes typically 1945, and Kendrew had just finished  Pioneers of the field to allow him to figure out how the needs a detailed picture of the serving as an advisor to Lord of x-ray crystallography, polypeptide chain is folded up molecular structure of the cavity into Mountbatten in Ceylon. Max Perutz and John into a compact ball. He published which it must fit with lock-and-key For another thing, Perutz had Kendrew won the Nobel this model 50 years ago this precision. never had a research student before. prize for chemistry in month,1 laying down a landmark Even though the structures But what troubled him most was 1962. At least eight in the histories both of molecular- of protein molecules with the the suspicion that working in Nobel prizes have been structure determination by x-ray complexity of myoglobin can now be his lab would be a very slow and won by researchers in crystallography and of structural solved in a matter of days rather than laborious way for Kendrew to get the field biology. years, structure determination is still a PhD. For Perutz was trying to use  Modern, automated Two years later Kendrew and a limiting factor in understanding x-ray crystallography to deduce and high throughput his colleagues had refined their how cells work. Today many of the the structures of proteins. The approaches to x-ray techniques sufficiently to see really difficult structures are those of arrangements of atoms in these crystallography myoglobin’s shape at a resolution vast multi-molecule complexes such biological molecules were evidently now mean that new of 2 Å (ångströms) – enough to let as the ribosome (the cell’s protein- fearsomely complicated, and not biomolecular structures them work out where some of the synthesis machine), which stretch even Perutz was sure they could be are decoded every week individual atoms are. This work, the techniques as far as they can truly deduced with beams of x-rays.  Very intense x-ray and the joint efforts of Kendrew go. The sheer number of different It is hard to imagine, Perutz said in sources are now enabling and Perutz on the structure proteins in the cell is prompting 1997, ‘how courageous Kendrew’s rapid structural changes of haemoglobin, as well as the the development of automated decision was then to take up protein in a protein, linked to its pioneering technical advances that methods that can churn out crystallography’. The reason he function and binding, to entailed, earned the two men the protein structures like production had no students was because be mapped over time Nobel prize in chemistry in 1962. plants; but many proteins refuse ‘responsible dons advised graduates Without achievements like these to form crystals good enough for against joining such a forlorn that revealed the three-dimensional diffraction techniques to work undertaking’. Right: a model of the atomic structures of proteins and well. Meanwhile, the emphasis is In the event it proved anything crystal structure of other complex biomolecules, starting to shift from structure to but forlorn, although the challenge myoglobin at the Science molecular biology could hardly exist. dynamics: to understand protein was every bit as difficult as Perutz Museum, London, UK A protein’s structure holds the key to function, we need to know not just 50 | Chemistry World | March 2008 www.chemistryworld.org

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what the molecules look like in a cracked. It involved making crystals static crystal, but how they change that contain very heavy metal atoms, shape, in the blink of an eye, as they such as uranium, which scatter x- perform their roles in the cell. rays strongly and thus provide a set of reference points in the diffraction Whale crystals pattern. If the pattern from heavy- When Kendrew arrived in his lab, atom crystals is compared to that Perutz was attempting to use x- from native crystals, the phase ray crystallography to solve the problem can be solved. structure of haemoglobin. The Haemoglobin contains sulfur technique, devised by the German atoms to which mercury ions physicist Max von Laue, had been could be conveniently attached. developed at Cambridge by William But myoglobin lacks convenient Bragg and his son Lawrence in the attachment sites for metal atoms, 1910s, who worked out the atomic and Kendrew and his colleagues structures of many inorganic had to resort to throwing all sorts of crystals. It relied on interference metals at the molecule until some between x-rays scattered from stuck. This allowed them to obtain MEDICAL RESEARCH COUNCIL LABORATORY OF MOLECULAR BIOLOGY, CAMBRIDGE, UK CAMBRIDGE, BIOLOGY, MOLECULAR OF LABORATORY COUNCIL RESEARCH MEDICAL different planes of regularly stacked three-dimensional structures. atoms, which created a pattern of Their 1958 paper showed pictures bright spots that could be recorded of the electron density in slices of on photographic film. From the the crystals – it’s the electrons that positions of these spots, one could actually scatter the x-rays – from calculate the positions of the crystal which the shapes of the folded planes, and thus where the atoms chains could be inferred. were situated. In the 1930s, J Desmond Bernal A brighter view and Dorothy Hodgkin in the It is a testament to the abiding Cavendish lab showed that protein importance of crystallography in crystals could yield sharp diffraction the sciences that at least eight Nobel patterns, raising the prospect that prizes have been awarded for work their structures could be decoded. directly relating to its development But it was already clear by then or use. Von Laue and the Braggs that proteins are large molecules were awarded in successive years, containing hundreds or thousands and just two years after Perutz and of atoms, and figuring out where Kendrew’s prize, Dorothy Hodgkin all the atoms sit would be daunting. horse myoglobin, but then he Above: John Kendrew was given the chemistry Nobel Perutz began to work as Bernal’s realised that diving mammals (left) and Max Perutz for her crystallographic studies of student in 1937, and a year later such as whales have much more with their model in biomolecules, including insulin and Lawrence Bragg became director myoglobin in their muscles, because progress vitamin B12. of the Cavendish. Perutz said that they need a big oxygen store. Below: a protein crystal For large molecules like proteins, Bragg’s enthusiasm for applying x- Perutz got hold of a large chunk of however, the difficulty is not just ray crystallography to proteins was sperm whale meat from Peru, and that there are so many atoms to vital to the success of an enterprise Kendrew was able to extract enough locate. Crystallography needs that needed patient support over myoglobin from this to make many years. ‘He was fascinated by beautiful, ‘sapphire-like’ crystals. the idea that the powers of x-ray But there was a problem. The analysis might be extended to the amplitude of the scattered x-rays giant molecules which form the is proportional to the brightness catalysts of living cells,’ Perutz of the spots in the pattern. But wrote. to calculate back from the At first, Perutz suggested that diffraction pattern to the Kendrew compare the diffraction location of the atomic planes, patterns of adult and foetal sheep it’s also necessary to know haemoglobin. Kendrew soldiered the phases of the waves: how on with this, but it just wasn’t their undulations are in or possible at that time to convert the out of step with one another. diffraction data to anything more This information just isn’t there than a rough outline of the protein in the diffraction photos. crystals, chains. (Perutz finally published the In 1953, Perutz saw but not all detailed structure, still at less than how this ‘phase proteins will easily atomic resolution after 22 years of problem’ form them. That’s effort, in 1960).2 Kendrew decided could be particularly a problem that he might have more success for membrane-bound with myoglobin, which is only a proteins, which have quarter the size of haemoglobin. ‘greasy’ surfaces compatible with The problem was to make crystals the fatty-acid lipids of membranes. large enough to diffract well. This makes them rather insoluble

MAX PLANCK WORKING GROUPS ON STRUCTURAL MOLECULAR BIOLOGY, HAMBURG, GERMANY HAMBURG, BIOLOGY, MOLECULAR STRUCTURAL ON GROUPS WORKING PLANCK MAX Kendrew couldn’t get them from in water. But such proteins typically 52 | Chemistry World | March 2008 www.chemistryworld.org

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stick out into the cytoplasm – and NATURE so they don’t generally dissolve  Oxford Diffraction is based well in organic solvents either. It’s in the UK. It was spun out of therefore hard to grow good crystals Oxford Instruments to develop, from them. manufacture and supply analytical A milestone in tackling this instrumentation for x-ray issue was the x-ray structure crystallography. determination of the bacterial www.oxford-diffraction.com photosynthetic reaction centre (PRC), which won the 1988 Nobel in chemistry for Johann Diesenhofer,  Bruker AXS is based in Robert Huber and Hartmut Michel. Karlsruhe, Germany. The company, Michel used small, soap-like which is part of Bruker, specialises surfactant molecules to solubilise in analytical x-ray systems and PRCs from photosynthetic purple complete solutions for material bacteria. The surfactants are analysis. One of its products, surrogates for membrane lipids, but the Microstar Ultra x-ray source instead of packing into continuous for biological crystallography, sheets they just cluster around the received an R&D 100 Award in fatty parts of the PRCs, creating 2007. micelle-like structures with their www.bruker-axs.de water-soluble heads at the surface. Michel managed to get PRC-  Molecular Dimensions is surfactant complexes to form nice a UK-based supplier of kits crystals, and he then collaborated and reagents for biomolecular with Diesenhofer and Huber to structure research. These include deduce the structure using Perutz’s ready-to-use matrices for crystal heavy-atom substitution to solve the growth screening and accessories phase problem.3 including vapour diffusion plates This work benefited from the and cover slips. availability of very intense x-ray www.moleculardimensions.com beams, in that case produced at the The predicted called the Structural Genomics DESY synchrotron in Hamburg. structure of a protein Consortium, based in Toronto,  Rigaku is a Japanese designer Such sources have been a boon for (grey) overlaid with Oxford and Stockholm, added of analytical and industrial protein crystallography, because its experimentally 50 such structures to the Protein instruments, including HomeLab more intense beams allow good determined structure. Data Bank. Last October the systems – combinations of x-ray diffraction patterns to be obtained consortium announced its 500th generator, optics, area detector, from smaller samples, reducing protein structure, an RNA helicase software and crystal cryo-cooling the need to make large, perfect linked to viral immunity. Even with system – each based around a crystals. In 1999, researchers at the projects like this, however, mapping specific x-ray source. University of California at Santa the protein landscape is a huge www.rigaku.com Cruz used the bright x-ray beam challenge. To understand protein of the Advanced Light Source at function and to plan drug design,  Oxford Cryosystems is the UK- the Lawrence Berkeley Laboratory it is often necessary to know the based designer and manufacturer in California to obtain the first structures not only of native proteins crystallographic structure of a but of many mutant forms too. of The Oxford Cryostream – the 4 very first commercially available complete bacterial ribosome. But But because the basic task is open flow nitrogen cooler for with a resolution of just 7.8 Å, it was always the same – make good crystallography. a fuzzy picture. By 2005, Jamie Cate crystals, collect the diffraction www.oxfordcryosystems.co.uk and colleagues at Berkeley, again pattern, and crunch the numbers working at the ALS, had sharpened until a ‘stable’ structure emerges this to 3.5 Å – which, coupled – it lends itself to automation.  Researchers can apply for with atomic-resolution data of There are now highly automated beamtime at the European individual ribosomal subunits, offers methods for ‘high throughput’ Synchrotron Radiation Facility in something like an atom-by-atom ‘If we could protein crystallography that produce Grenoble, France and the Institut view of how this protein-synthesis understand the and analyse hundreds of crystals Laue-Langevin which shares the machine works. a week. Some of these operate on same site and currently provides rules that lead an industrial scale using robotic the most intense neutron source Quick snaps from protein preparation procedures, and they in the world. The new Diamond Protein structures are now routinely rely on synchrotron sources to synchrotron in Oxfordshire, UK solved at a rate of more than one a sequence to gather enough data for structure also has seven beamlines now in week. Many are bacterial proteins, shape, we determination in a very short time, operation. since these tend to be simpler and using crystals that can be as small www.esrf.eu thus easier to purify and crystallise might not as ten micrometres across.6 The www.ill.fr than human proteins. But proteins need protein diffraction patterns can be analysed www.diamond.ac.uk relevant to human disease are by computers, but it is hard to leave gradually giving up their secrets crystallography everything to machines: choosing – in 2005, for example, a project at all’ good crystals is an art that generally 54 | Chemistry World | March 2008 www.chemistryworld.org

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depends on human judgement. The small crystals are also often fragile, making them difficult to manipulate with robotics. In his 1988 Nobel lecture, Robert Huber pointed out that one of the biggest mysteries of protein structure is how they fold from an extended polypeptide chain into a functional, compact three-dimensional shape. ‘An ultimate goal for which we all struggle is the solution of the folding problem,’ he said. If we could understand the rules that lead from protein sequence to shape, we might no longer need protein crystallography at all: in principle, a protein’s shape could then be deduced simply from the sequence of the gene that encodes it. But to understand protein folding we need to follow the process A micrograph (above) from moment to moment: it is a and diffraction pattern question not of structure but of taken by ultrafast dynamics. At face value, diffraction electron microscopy and might appear to be useless for that, diffraction developed because it relies on the existence at Caltech: pulses of of static structures in a crystal. electron beams obtain But not necessarily. If we could frames as a function of obtain a diffraction pattern fast time enough, we might be able to work out the instantaneous structure of an evolving molecular shape, and thus follow the trajectory it takes. That means collecting lots of data literally in a flash. But this is now becoming feasible.7 Late last year, for instance, Ahmed Zewail’s group at the California Institute of Technology in

Pasadena reported a ‘four- ZEWAIL AHMED dimensional’ structure of a vanadium dioxide simulations. But current machines of volunteers. crystal as it undergoes simulation methods just Even if a solution to the folding a phase transition. The aren’t fast or accurate problem is going to render protein fourth dimension is time: enough to follow folding crystallography redundant, then, it using ultrashort pulses of all the way from start to looks set to rely on the achievements electrons, Zewail’s team could end. David Baker at Stanford of structure determinations past. follow atomic rearrangements by University in California has As Huber puts it, ‘it is certain that electron diffraction on timescales of developed a method in which the end of protein crystallography just a few picoseconds.8 In principle, simulations draw on the knowledge will only come through protein such methods might also reveal gained from previous protein- crystallography.’ the changes that occur as proteins structure determinations, using bind their targets: in contrast to the sequence matches between the Philip Ball is a freelance science writer classic ‘lock-and-key’ idea, these ‘Ultrashort protein being studied and those based in London, UK events rely on a lot of floppiness and in data banks, to provide good x-ray pulses References flexibility in the binding sites, which guesses about how parts of the 1 J C Kendrew et al, Nature, 1958, 181, 662 could supply key clues to making could map chain will fold. Last year, Baker’s 2 M F Perutz et al, Nature, 1960, 185, 416 protein-binding drugs.9 Researchers team reported that their so-called 3 J Diesenhofer, et al, Nature, 1985, 318, 618 at the ALS are now aiming to protein motions Rosetta technique could correctly 4 J H Cate et al, Science, 2001, 285, 2095 5 B S Schuwirth et al, Science, 2005, 310, 827 create ultrashort x-ray pulses on timescales predict the structure of a 112- 6 B Rupp, in Proteomics, ed. A. Edwards that might enable the mapping of residue bacterial protein from its (Marcel Dekker, 2003) protein motions on timescales of of attoseconds sequence.10 This is still a small 7 K J Gaffney and H N Chapman, Science 2007, attoseconds – billion-billionths of a – billion- protein, however, and even then the 316, 1444 second. task was computationally expensive, 8 P Baum et al, Science, 2007, 318, 788 billionths of a 9 D Bourgeious and A Royant, Curr. Opin. Struct. Another approach to the protein- relying on distributed computing Biol., 2005, 15, 538 folding problem is to use computer second’ that scavenges spare time from the 10 B Qian et al, Nature, 2007, 450, 259 56 | Chemistry World | March 2008 www.chemistryworld.org

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