DOI: 10.1126/science.1247829, 1102 (2014);343 Science

Elspeth F. GarmanBiological MacromoleculesDevelopments in X-ray Crystallographic Structure Determination of

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phasing algorithm, utilizing the fundamental knowl-edge that electron density in a crystal structure mustbe positive. The method has rapidly become apopular alternative for data sets where traditionalmethods fail (68). CFA has amajor advantage overtraditional solution methods, as the space group ofthe structure does not need to be determined beforeuse. It is the only structure solution method that iscurrently extensible to systems where the full sym-metry of the system is described by3+ndimensions.

The FutureChemical andmaterials sciences lie at the basis ofthe next generation of smart materials, fabrics,and devices, and x-ray crystallography is funda-mental to their design and successful application.The use of crystallography in online analysis willcontinue to be an essential industry tool, and in-struments will become faster, smaller, more por-table, and applicable in the field for importanthealth problems in remote areas and the devel-oping world. Concurrently, the development ofnew powerful x-ray sources for the laboratory, aswell as at global central facilities, will enable newdiscoveries at higher resolution by using muchsmaller crystals, and importantly, these experi-ments will use much less of the crystalline mate-rials in the studies, whether pharmaceuticalcompounds, precious metals, or the rare chem-icals that are needed in modern electronics.Recent discoveries at the molecular level forsmart materials with clever magnetic and elec-trical properties (e.g., single-molecule magnets)require extensive dynamic structural studies toexplain the subtle molecular changes under appliedexternal fields so that these changing propertiescan be exploited in the next generation of devices.Taking crystallography to other planets, most re-cently Mars, has challenged the imagination ofcrystallographers, engineers, mathematicians, andmany other materials scientists, with staggeringresults, and we can expect to see more missionsthat take remote-controlled laboratories to distantplacesmissions that were unimaginable a fewyears ago.Thecollaboration of scientists developingportable x-ray sources, fast, sensitive detectors,intelligent robots, innovative software, and dataanalysis methods will find many applications andchallenges for crystallographers in the decadesahead. Fortunately, crystallography has a long his-tory of sharing ideas, experiences, expertise, methods,and software for the common good (69, 70).

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Biochem. Soc. Trans. 41, 12601264 (2013).

Acknowledgments: We are indebted to our colleagues inDurham and elsewhere, who use these techniques routinelyand who have read the manuscript and helped to providereferences that we might have missed. We are grateful also tothe reviewers for comments on the manuscript.

10.1126/science.1247252

REVIEW

Developments in X-ray CrystallographicStructure Determination ofBiological MacromoleculesElspeth F. Garman

The three-dimensional structures of large biomolecules important in the function and mechanistic pathwaysof all living systems and viruses can be determined by x-ray diffraction from crystals of these moleculesand their complexes. This area of crystallography is continually expanding and evolving, and the introductionof new methods that use the latest technology is allowing the elucidation of ever larger and more complexbiological systems, which are now becoming tractable to structure solution. This review looks back at what hasbeen achieved and forward at how current and future developments may allow technical challenges to be overcome.

Macromolecular crystallography enablesthe three-dimensional (3D) structuresof large biologically interesting mole- cules to be determined. Structures of proteins andnucleic acids determined bymacromolecular crys-tallography are vital for elucidating protein function7 MARCH 2014 VOL 343 SCIENCE www.sciencemag.org1102

and intermolecular interactions and for improvingour understanding of basic biological and bio-chemical mechanisms and disease pathways. Theirimmediate practical application is in the design ofpharmaceuticals, in which they play a central rolein drug discovery.

This branch of crystallography has dramati-cally advanced over the past 80 years since the 1934initial observation of diffraction from crystals of asmall protein, pepsin, and the first protein struc-

ture determination (myoglobin) (Fig. 1A) in 1958.Haemoglobin followed, and then in 1965 the firstenzyme structure, lysozyme (Fig. 1B), was solved.The recent characterization of the entire ribosome(Fig. 1C) revealed one of the essentialmachines oflife, comprising a vast complex of molecules con-sisting of ~280,000 nonhydrogen atoms: more than2.5 orders of magnitude larger than the 1260 inmyoglobin. The field has been awarded 28 NobelPrizesstarting with father-and-son teamWilliamHenry and (William) Lawrence Bragg in 1915with the latest being the 2012Chemistry PrizewonbyKobilka andLefkowitz for studies onGproteincoupled receptors (GPCRs), crucial cellular sensors

for signaling proteins and hormones. These NobelPrizes signal the effect that crystallography has hadand continues to have in the world of cutting-edge research.

Macromolecular crystallography was born withthe pivotal discovery by Bernal and Crowfoot (1)that pepsin crystals retained their order if kept hy-drated in a capillary tube sealed at each end duringx-ray diffraction experiments. Unlike the crystalsformed by inorganic or small organic compounds,macromolecular crystals can contain up to 90% sol-vent surrounding themolecules. The intermolecularinteractions supporting the crystalline lattice areweak. The success of diffraction experiments

Department of Biochemistry, University of Oxford, South ParksRoad, Oxford OX1 3QU, UK.

E-mail: elspeth.garman@bioch.ox.ac.uk

Stroma

Lumen

7.0 nm

A B

C D7.5 nm

Fig. 1. Visualization ofmacromolecular structures. (A) Balsa wood modelof myoglobin at 5 resolution (45) and a model of a monoclinic crystal, madeby H. Scouloudi, 1969. (B) Wire model of lysozyme structure (39). Modelconstructed by W. Browne and M. Pickford circa 1965. Refurbished by A. Toddand Unicol Engineering of Headington, Oxford, UK. Blue, nitrogen; red, oxy-gen; black, carbon; yellow, sulfur; and gray, hydrogen bonds. (C) Ribosome70S particle at 3.5 resolution (46). 30S subunit and tRNA, PDB entry 2wdk;

50S subunit, PDB entry 2wdl. The 30S subunit is shown in purple (pale forprotein, dark for RNA) and the 50S subunit in blue (pale for protein, dark forRNA). The tRNA is in gold. Figure made with CCP4mg (47). (D) Photosystem IIat 1.9 resolution. PDB entry 3arc (48). The protein is shown in blue and thechlorophylls in green. The oxygen-evolving cluster is depicted as spheres andhighlighted by dotted circles, and the membrane bilayer is indicated by ashaded box. Figure made with CCP4mg.

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critically depends on crystalline order, which usu-ally deteriorates if the crystals are allowed to de-hydrate. Many of the technical challenges in thefield arise from this property of protein crystals.

Crystallographic macromolecular structures aretime and space averages over the many millionsof macromolecules within the crystal. A largeprotein crystal is typically smaller than 100 mmin all three dimensions. For an average-sized 5-nm-diameter globular protein, such crystals wouldcontain ~1013 molecules. The dynamical behav-ior of the molecules within a crystal allows only alimited sampling of the conformational space ofthe protein because the crystallization conditionsbias the behavior. Better information on dynamicalproperties is required to fully understand protein-protein interactions and pathways. Techniques toaddress this issue are being explored with the aidof newly available technology, and current ap-proaches are described elsewhere in this issue (2).

For the past 20 years, over 95% of macromo-lecular structures have been determined from crys-tals held at cryotemperatures (~100 K) because therate of radiation-induced damage is lower by afactor of ~70 comparedwith room temperature (3).Although 100 K is far from physiologically rele-vant temperatures, it is clear from structural studiesof the same proteins at different temperatures thatthe overall fold of the alpha-carbon amino acidchain is temperature independent. More orderedwater molecules can be located in structures deter-mined at cryotemperatures, and alternative confor-mations of side chains tend to be better defined.This is because the dynamic disorder in the proteinis frozen out and the observed substate popula-tions reveal only the static disorder. Because thesedetailed observations are not necessarily physio-logical relevant, ideally structures would also bedetermined at room temperature if this could beconveniently expedited.

Currently, some promising new developmentsin macromolecular crystallography are unfolding.Future growth areas summarized below are mem-brane protein crystallography, and room-temperaturedata collection both at synchrotrons and at the re-cently introduced x-ray free-electron lasers (XFELs).

The PipelineThe deployment of new technology and meth-odology is continually streamlining the pipelineinvolved in macromolecular structure solution(Fig. 2) and improving the success rates forchallenging cases. However, the major bottleneckremains the growth of diffraction-quality crystals.

Before crystallization canbe attempted, sufficientquantities of protein must be purified, usually asrecombinantmaterial frombacterial, yeast, insect, ormammalian cells. Expression systems have becomehigh throughput as a result of more rapid and reli-able cloning tools and the more widespread use ofautomation and bioinformatics. These developmentspermit better-informed and extensive screening ofexpression vectors, protein sequences, and hetero-

logous host cells (4). It can still be a labor-intensiveand time-consuming task to optimize the system toproduce enough protein for crystallization trials.However, with recent methodological progress, thestructures of an increasing number of proteins thatwere historically viewed as challenging (e.g., mem-brane proteins, posttranslationallymodified proteins,and protein complexes) are now being solved.

An important development has been the useof autotrophic strains for the incorporation ofseleno-methionine into recombinant protein, be-cause the selenium allows the structure to beexperimentally phased by the multiwavelengthanomalous dispersion (MAD) method (5).

To maximize the chances that crystals will grow,the protein must be as homogeneous and pure aspossible, so itmust usually be in a single oligomericstate. Large losses of protein may be experiencedduring purification, but this step is vital for successfulcrystallization. Techniques for assessing protein pu-rity have advanced considerably, and a variety ofmethods are now used, including dynamic lightscattering and coupling of size-exclusion chroma-tography with multiangle laser light scattering.These reveal whether a protein sample is mono-dispersed and homogeneous, often giving a goodindication as to whether it might crystallize.

Although the parameters governing the pro-cess of protein crystallization are now better un-derstood through research into crystallogenesis, itis not yet possible to predict the conditions underwhich a particular protein will crystallize. Thus, theapproach is still to coarse-screen a wide range ofchemical conditionssuch as buffer type, tem-perature, pH, protein concentration (typically 10 to20 mg/ml), cocktails of detergents if it is a mem-brane protein, precipitants (organic solvents, salts,and polymers), presence or absence of divalentcations, and additivesin the hope of obtaining afewhits. Screeningon a finer grid that samples aroundthese promising conditions then allows optimiza-tion, which may result in diffraction-quality crystals.

Crystallization robots that can routinely dis-pense low-volume drops (as low as 50 nl protein +50 nl of precipitant solution) permit thousands ofconditions to be coarse-screened. This has greatlyincreased the likelihood of crystallization condi-tions being found given limited protein volumes;for instance, with 150 ml of protein, ~1500 trialdrops of 100 nl + 100 nl could be tested in slender96-well plates holding two conditions per well.Larger volume than the minimum 50 nl is usuallydispensed, because scaling up crystallizationconditions from such small drops can be proble-matic due to changes in surface-to-volume ratios.The trays are typically kept at a constant temper-ature (e.g., 4C or 20C) in crystal hotelsequipped with imaging devices that automaticallyphotograph the crystallization drops at regular in-tervals, and these images can then be scored usingautomated crystal recognition software. Thus,muchof the drudgery has been removed from the searchfor suitable conditions. The successful development

of such automated systems owes much to theinvestment of resources and timemade in structuralgenomics centers in the early part of this century.

Once a crystal has been obtained, it must usuallybemanually harvested from its growth drop before

I F (protein)

Overexpression/produce pure

protein

Crystals . . .

Molecularreplacement

Derivatization/Se-Met

Solvephases SIR/MIR/MAD/

SAD

Initialstructure

Iterative refinement

Characterization/Quality/

Validation

Diffraction,I Resolution

RCSB

Fig. 2. Diagramshowing, fromtoptobottom, thepipeline for macromolecular structure solution.

7 MARCH 2014 VOL 343 SCIENCE www.sciencemag.org1104

being irradiated with x-rays. Successful vitrifica-tion (Fig. 3) of the crystal for data collection atcryotemperatures generally requires the presenceof cryoprotectants. The flash-cooling of crystals(6), held in cryoloops by surface tension, is a stepin the macromolecular crystallography pipelinethat has so far proved difficult to automate. Com-mercial cryoloops are available in a range of sizesand made from rayon, microfabricated polyimidefilm, and etchedmylar, somehaving integralmeshesto support fragile crystals or many small crystalssimultaneously. Technically, there is a pressing needfor automatic crystal harvesting and sample handl-ing methods to overcome this pipeline bottleneck.

The evolution of storage ring sources to thecurrently available third-generation synchrotronsources (7) (Fig. 4) in conjunction with fast andaccurate x-ray detectors has revolutionized mac-romolecular crystallography for the collection ofdiffraction data. The very high synchrotron sourceflux densities (photons per s permm2) allowweaklydiffracting or smaller crystals to be used for structuredetermination. They provide parallel and stablebeams, many of which can be tuned to deliver inci-dent x-ray energies from 6 keV to 20 keV (~2.1 to0.62 ), giving access to the absorption edges of awide range of metals for experimental phasing bythe MAD method. Pioneering beamlines suitablefor data collection at longerwavelengths (up to 4)are under construction to enable more experimentalphasing of structures using the anomalous signalfrom intrinsic sulfur atoms in proteins. The nowrobust top-upmode at synchrotron sources, inwhichthe storage ring is continuously fed with electrons,results in stable experimental conditions for longperiods of time. Detector technology has moved onapace, driven by the requirement for faster and largerposition-sensitive devices. Originally, thefield used photographic film and proportio-nal counters, and then position-sensitivemultiwire gas-filled detectors, adapted tele-vision tubes, imaging plates (reusable film),charge-coupled device detectors, and, mostrecently, pixel detectors (8).

Most synchrotron beamlines are cur-rently equipped with sample-mounting ro-bots that transfer crystals from a liquidnitrogen Dewar to the goniometer into astream of 100 K nitrogen gas, meanwhilekeeping them cryocooled. The increasedreliability of these robots has led to re-mote data collection in which crystals aredelivered to the beamline and the researchercontrols the beamline hardware remotely.Synchrotron beamline availability is nowsuch that many in-house systems are beingdecommissioned.

A number of synchrotron beamlines arenow providing particular special facilities,such as microfocus beams (diameters downto 1 mm).With the necessary supporting soft-ware, these beams can be used to map thediffraction properties of a crystal so that the

best place for data collection can be selected. To min-imize background andmaximize the signal-to-noiseratio, the beam and crystal size should be matched.Thus, these microbeams are ideal for use with mi-crocrystals, where many crystals can be mountedon one loop and then individually irradiated.

Additional instruments have beenmade avail-able to augment the information that can be ob-tained from crystals through simultaneous datacollection using complementary techniques. Forexample, most synchrotrons now have a beam-line onto which amicrospectrophotometer can bemounted, which can provide valuable data onredox protein states and radical formation duringx-ray irradiation (9). Another useful new additionis a device to carry out on-line controlled dehy-dration of protein crystals (10), because in somecases this technique can improve the diffractionquality in a reproducible way. For instance, F1adenosine triphosphatase crystals were improvedfrom 6.0 to 3.84 resolution by dehydration (10).

Automated data reduction pipelines are nowwidely available atmost beamlines, and these allowon-line evaluation of the results so that more datacan be collected immediately if necessary, sub-stantially improving the outcomes of the experi-ment. However, even for cryocooled crystals, theage-old problem of radiation damage remains anissue and can result in failed structure solutiondue to the degradation of diffraction quality andthe onset of specific structural damage (11) be-fore enough data have been obtained. Research isongoing to understand the variables involved andto seek mitigation strategies (12). The extent ofdamage at cryotemperatures is proportional to theabsorbed dose, and an experimental dose limit of30Mgy, beyondwhich structural informationmay

become compromised, has been determined (13).Software (Raddose-3D) is available to model 3Ddose profiles for a range of experimental strat-egies (standard, helical, and translational). Thesesimulations can be used to plan experiments thatresult in more homogeneous dose distributions,reducing the extent of differential radiation damageacross the sample and improving data quality (14).

A number of streamlined packages are availableto analyze the diffraction data and to reduce them toa unique set of reflections so that structure solutioncan commence. Concomitant with the developmentsin hardware and the automation of data collection,computational tools for structure solution have seenhugeprogress over thepast decade.Crystallographicsoftware, such as that distributed by CollaborativeComputational Project Number 4 (CCP4) (15) andPHENIX (16), can now solvemany structures with-out human intervention, fromdata reduction throughphasing and electron density map calculation, mapinterpretation (model building), structure refinement(completion), and deposition in the Protein DataBank (PDB). For the cases in which automatedsolution is still not possible, the software is betterable to analyze the pathologies causing it to failand to guide the crystallographer to a manual solu-tion.Molecular replacement can now succeed withvery distant models or even secondary structureelements, as implemented in Phaser (17) andArcimboldo (18). Experimental phasing can nowsucceed with very weak anomalous signals dueto progress in phasing software [e.g., the SHELXsuite (19)] and improved methods to enhance theanomalous signal when combining data collectedfrom a large number of different crystals [e.g., (20)].

After an initial model is obtained, the structuremust be refined to optimally match the model to the

electron density. This process is fast andhas a wide radius of convergenceforexample, in Phenix.refine (16) and Refmac(21). Software for automatically buildingatomic models into electron density mapsis increasingly more robust, and for man-ual building, programs such as Coot (22)tremendously aid the iterative process ofmodel refinement and rebuilding. Thegraphical capability now available allowsmacromolecules to be represented muchmore speedily, cheaply, and convenientlythan with balsa wood and wire models(Fig. 1, A and B). For the last step in thepipeline, convenient new tools are alsoavailable for the validation of the geom-etry and quality of structures before sub-mission of atomic coordinates to thePDB (23).

Future Growth AreasCurrent growth areas in which macro-molecular crystallography is likely to haveconsiderable future impact include mem-brane protein structure solution, renewedinterest in room-temperature structure

B

A

20m

Fig. 3. Macromolecular crystals ready for data collection. (A)Cryocooled 0.5-mm-sized crystal of Salmonella typhimurium neuramin-idase in a 20-mm-thick rayon fiber cryoloop held in a 100 K nitrogen gasstream. The transparent film of solid cryobuffer supporting the crystalindicates that no crystalline ice has formed that could interfere with thecrystal diffraction pattern. (B) In situ data collection from bovine entero-virus crystals; despite the rapid and dramatic disruption of the crystallattice, small amounts of high-quality data can be collected in a serialmanner until a complete data set is obtained (30). Reproduced bypermission of the International Union of Crystallography (IUCr).

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determination at synchrotrons, and the possibil-ities offered by XFEL x-ray sources.

About 30% of the proteins coded by the hu-man genome are membrane proteins. Determiningthe structure of these represents a major challengefor conventional techniques, because the crystalli-zation step usually relies on controlled dehydrationof a solution of protein. Because proteins extractedfrom the membrane are by their very nature insol-uble in aqueous systems, new methods have to beemployed to obtain crystals; the proteins mustnormally be solubilized in detergents, both throughoutpurification from cell lysates and during crystallization.This greatly increases the number of variable crystal-lization parameters to be explored and makes thesearch for suitable conditions both time-consumingand expensive. The addition of detergents is proneto destabilize the protein, and much trial and erroris required for successful outcomes. As a result,out of 97,362 protein structures (as at 28 January2014) deposited in the PDB, thereare only 1394 membrane proteinstructures (24), although the num-ber is increasing rapidly. In partthis is due to the development andsuccess of a new crystal-growingtechnology: the in meso method,which makes use of lipidic meso-phases and is also referred to as thelipid cubic phase (LCP) method.This uses monoolein, which has awell-characterized phase diagram ofcomposition (water/lipid) againsttemperature (25). Crystallization ro-bots to dispense LCP are now avail-able, and they substantially simplifyand accelerate the setting up ofscreens. However, safe removal ofcrystals from LCPmaterial requiresskill and patience on the part of theexperimenter, so this stage is ripefor further innovation. On contactwith air, the LCP can swiftly de-hydrate unless additional crystalli-zation solution is added, and it alsobecomes opaque and birefringent,making it hard to locate and to har-vest the crystals. Once in a cryoloopand flash-cooled (no added cryo-protectant is needed) for cryodatacollection, the LCP again often be-comes opaque, and any crystalswithin it become invisible. The auto-mated grid scans of the x-ray beamover the loop area to detect crystaldiffractionabovehave alleviated thisproblem, andwork to image suchcrys-tals by x-ray microradiography andmicrotomography is ongoing (26).

Membrane protein crystals grownin cubic and sponge phases haveyielded data revealing, for example,the structural basis for the counter-

transport mechanism of a H+/Ca2+ exchanger (27)and the structure of the 2 adrenergic receptorGproteinactive complex (28), a GPCR in associa-tionwith its cognateGprotein.Correct functioningof GPCRs is vital for our senses of smell, taste,and sight and is also involved in almost all signalingprocesses, including cellular responses to neuro-transmitters and hormones. Because roughly halfof all modern drug targets are GPCRs, theirstructural elucidation is one of the major high-lights of recent research.

The ability to crystallize membrane proteins ina membrane-like environment such as LCP opensthe possibility of gaining more biologically rele-vant information on protein-lipid interactions. Suchinteractions help regulate subcellular localizationand determine the activities of transmembraneproteins, yielding, for instance, insight into thefunction of the receptor tyrosine kinase family.These proteins are implicated in the progression

of many types of cancer, as well as being vitalregulators of normal processes in the cell (29).

In the search for suitable crystallization condi-tions for membrane proteins, it is often highlyinstructive to test the diffraction properties of puta-tive crystals obtained from a coarse crystallizationscreen. This necessity has prompted beamlinescientists at a number of synchrotrons to adaptconventional goniometers so that entire 96-wellcrystallization plates can be mounted in the x-raybeam and translated to enable irradiation of in-dividual wells containing putative crystals. In somecases, a limited rotation capability has also beenincorporated into the beamline hardware and soft-ware, so that complete ensemble data sets consti-tuted of images from many crystals can now becollected and can result in successful structuresolution (30), without the necessity for any post-growth handling of crystals. Figure 3 shows a crys-tal of bovine enterovirus at room temperature in a

Fig. 4. Progression of hardware for macromolecular crystallography experiments. (A) A Hilger-Watts lineardiffractometer as used to collect the data used to solve the structure of lysozyme in 1965 (49). (B) The first third-generation synchrotron x-ray source: the European Synchrotron Research Facility (ESRF), Grenoble, France. Photocourtesy of ESRF/Morel. (C) Part of an XFEL: a 132-m-long undulator at the Linear Coherent Light Source, Stanford, CA,USA. [Photo courtesy of SLAC National Accelerator Laboratory, Archives and History Office]

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crystallization tray being consecutively irradiatedfor 0.5 s at four different positions by translatingthe tray before radiation damage effects causethe disintegration of the recently irradiated part.The success of this strategy relies heavily on thehigh speed of data collection and on the advent ofextremely fast pixel array x-ray detectors (PADs)(31). These are replacing the charge-coupled de-vice detectors that have been the macromolecularcrystallography workhorses for the past 10 years.

Currently, the biggest PAD is 425 by 435 mm2

and has a readout of 0.995 ms, a maximum framerate of 100 per second, and 6 million pixels. ThePAD readout times are so fast that they have re-sulted in a paradigm shift in the way the diffractionexperiment is carried out, with shutterless data col-lection becoming the norm: It is now unnecessaryto oscillate the crystal over a limited angular range(~0.1 to 1) and then close the shutter duringdetectorreadout. This change in experimental approachcombined with the high PAD frame rates dramati-cally increases the rate at which data can be col-lected, while concomitantly reducing demands onbeamline components such as x-ray shutters.

Experiments using a high-speed PAD havedemonstrated that it may be possible to collect dataat room temperature so quickly that the catastrophiceffects shown on Fig. 3B can at least partially beoutrun (32). There was already anecdotal evi-dence from early macromolecular crystallographysynchrotron experiments 30 years ago that room-temperature crystals lasted much longer than hadbeen expected, and during the past 5 years therehas been some debate as to the existence of a room-temperature dose-rate effect on radiation damageprogression. It would be most instructive to under-stand the details of the radiation chemistry pathwaysin room-temperature protein crystals during x-rayirradiation, so that the application of recent tech-nological developments could be optimized.

In conjunction with the in situ tray irradiationdescribed above, the opportunity to collect moreroom-temperature diffraction data by collecting itfaster has opened up the potential for proteinstructures to be determined with no postgrowthhandling being necessary. This is particularly perti-nent for virus crystals for which biological contain-ment requirements complicate traditional datacollection methods, but it is also important forsamples that prove difficult to handle or manipulateand for those that cannot be cryocooled withoutserious degradation of their diffraction properties.

Hardware developments for macromolecularcrystallography have not been confined to the im-provement in the size and accuracy of x-ray de-tectors. Since the early days of sealed-tube x-raysources, crystallographers have exploited the latesttechnical advances to obtain brighter beams.The huge increase in source brilliance (B) (mea-sured in units of photons per second per mm2 permillisteradian per 0.1% bandwidth, here called U)available today has been achieved through steadyprogress that has encompassed rotating anode

x-ray generators with magnetic liquid rotary vac-uum seals (B > 107 U), focusing optics fabricatedfrom alternating graded layers of high and lowatomic number elements (B > 108 U), synchrotron-fed electron storage rings equipped with bendingmagnets (B > 1010 U), wigglers (B > 1011 U), andthen ultimately in-vacuum undulators (B> 1012U),and finally the recent advent of XFELs at Stanford[Linear Coherent Light Source (LCLS)] (Fig. 4),SPring8AngstromCompactElectron-Laser (SACLA),and Deutsches Elektronen-Synchrotron (DESY)[Free Electron Laser Hamburg (FLASH)]. Forexample, the macromolecular crystallography CXI(coherent x-ray imaging) beamline at the LCLS istypically operated at 10 to 120Hz,with x-ray pulsesof around 1012 photons in a 10-mmfocus,which canbe tuned from70 to 300 fs at energies of 4 to 10keV(Bpeak > 10

33 U; Baverage > 1021 U).

Serial femtosecond crystallography (SFX) is atechnique in which protein nanocrystals suspendedin a liquid jet are streamed using a surrounding gasjacket (33) perpendicular to the beam direction sothat the x-ray pulses hit them to produce diffractionstills. These patterns are recorded on special PADdetectors (34). Typically, hundreds of thousandsof images are collected, a small fraction of whichshow a diffraction pattern, and a small percentageof these are suitable for structure solution. Thecollection of one still image per nanocrystal presentsa major challenge for available diffraction analysissoftware. In an ongoing effort, new methods (e.g.,Monte Carlo integration) are being employed toextract useful information from the many tera-bytes of data collected during every XFEL run.

Notable SFX results so far include the struc-tures of Cathepsin B (35) and photosystem I (36),bothdeterminedby themolecular replacementmeth-od. In another highlight, a combined spectroscopicand crystallographic study gave insights into theworkings of Photosystem II (37), a large complex oftransmembranemolecules (Fig. 1D), vital to photo-synthesis and thus to aerobic life. In late 2013, aproof of principle de nuovo structure determinationof soaked lysozyme nanocrystals (

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Acknowledgments: I am grateful to I. Carmichael, J. Helliwell,R. Ravelli, and the referees for their constructive comments onthis review; to M. Caffrey, J. Endicott, M. Higgins, A. McCoy,S. Newstead, and R. Owen for providing expert input; and toE. Lowe, J. Brooks-Bartlett, and J. Rowntree for their help with figures.

10.1126/science.1247829

REVIEW

Femtosecond Crystallography withUltrabright Electrons and X-rays:Capturing Chemistry in ActionR. J. Dwayne Miller1,2

With the recent advances in ultrabright electron and x-ray sources, it is now possible to extendcrystallography to the femtosecond time domain to literally light up atomic motions involvedin the primary processes governing structural transitions. This review chronicles the development ofbrighter and brighter electron and x-ray sources that have enabled atomic resolution to structuraldynamics for increasingly complex systems. The primary focus is on achieving sufficient brightness usingpump-probe protocols to resolve the far-from-equilibrium motions directing chemical processes that ingeneral lead to irreversible changes in samples. Given the central importance of structural transitions toconceptualizing chemistry, this emerging field has the potential to significantly improve ourunderstanding of chemistry and its connection to driving biological processes.

Chemistry has long been appreciated to bea race against time. One wants to createconditions to drive the desired chemistryfaster than other possible reaction routes. To thisobjective, we have been left to imagine the rela-tive atomic motions that lead the system throughan activation or energy barrier to convert to newchemical species. This conceptualization of chem-istry represents a classic thought experiment thatprovides the unifying language connecting thedifferent disciplines in chemistry as well as pro-

vides the conceptual bridge between biology andchemistry. The challenge is to depict transition-state structures that are taken to be energeticallyat the halfway point along an assumed reactioncoordinate connecting reactant and product states.This exercise is a useful pedagogical tool becauseit emphasizes the connection between the structureat critical transition points and barrier heights. Weneed this structural connection in order to properlythink about means to control barrier heights andthereby the chemistry (and biology) of interest.This practice can be justified for few atom systemsbut is questionable for most systems of chemicalinterest. For a molecule of N atoms, there are onthe order of 3N degrees of freedom or dimensionsto the problem to track all possible nuclear con-figurations. Imagine trying to map a surface with

hundreds of dimensions to give you all the routesinterconnecting different possible stability points.It would be extremely difficult to find general fea-tures for trekking between one stable valley, orstructure, to another.Here, one has tomarvel at chem-istry.Within the classic description of transition-stateprocesses, each molecule would have a distinctmany-body potential energy surface, with distinctmodes reflecting the different degrees of freedomneeded to describe the nuclear fluctuations. Eachdifferent molecule should be a new adventure;yet, chemistry involves widely applicable reactionmechanismsthat is, transferable concepts.

The problem to date is that we have been un-able to observe the key modes involved in directingchemistry. We have a very detailed understandingof equilibrium fluctuations of molecular systemsbased on vibrational spectroscopy as well as a hostof other experimental and theoretical methods.However, until recently there has been no directmeans to observe the primary atomic motions in-volved in structural transitions. With the recent ad-vances in ultrabright electron and x-ray sources, itis now possible to light up the atomic motions(via diffraction) on the prerequisite time scale toobserve the key modes governing chemistry (1).

Making Molecular MoviesTo get some appreciation of the experimentalchallenges, consider trying to build a camera tocapture atomic motions on the fly, to make amolecular movie. What is the shutter speed re-quired to follow chemically relevant atomic mo-tions? If we use the case of bond breaking, the timescale involved is the time it takes two atoms to movefar enough apart so that the interatomic potentialis no longer binding within kBT (where kB is theBoltzmann constant and T is the temperature).

1Atomically Resolved Dynamics Division, The Max Planck In-stitute for the Structure and Dynamics of Matter, The HamburgCentre for Ultrafast Imaging, Luruper Chaussee 149, Hamburg22761, Germany. 2Departments of Chemistry and Physics,University of Toronto, 80 St. George Street, Toronto M5S 1H6,Canada.

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