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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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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: [email protected]
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.
www.sciencemag.org SCIENCE VOL 343 7 MARCH 2014 1103
SPECIALSECTION
-
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).
www.sciencemag.org SCIENCE VOL 343 7 MARCH 2014 1105
SPECIALSECTION
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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]
7 MARCH 2014 VOL 343 SCIENCE www.sciencemag.org1106
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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.
7 MARCH 2014 VOL 343 SCIENCE www.sciencemag.org1108