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Archives of Biochemistry and Biophysics 602 (2016) 32e47

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Serial femtosecond crystallography: A revolution in structural

Jose M. Martin-Garcia a, b, Chelsie E. Conrad a, b, Jesse Coe a, b,
Shatabdi Roy-Chowdhury a, b, Petra Fromme a, b, *
School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604, USA
Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-7401, USA

a r t i c l e i n f o a b s t r a c t

Article history: Macromolecular crystallography at synchrotron sources has proven to be the most inuential method
Received 19 October 2015 within structural biology, producing thousands of structures since its inception. While its utility has been
Received in revised form instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as
16 March 2016
the need for large, well-diffracting crystals, and radiation damage that can hamper native structural
Accepted 21 March 2016
Available online 30 April 2016
determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the
emerging eld of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion
upon existing crystallographic constraints, allowing structural biologists access to previously restricted
X-ray free electron lasers
scientic territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femto-
Serial femtosecond crystallography seconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets
Protein crystallography comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orien-
Nanocrystals tations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation
Membrane proteins damage, even at room temperature, can now be achieved. This emerging eld has cultivated new
methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges
within SFX are reviewed herein.
Published by Elsevier Inc.

1. Introduction Wilhelm Conrad Ro entgen which catalyzed a revolutionary change

in our understanding of the physical world. The ability of X-rays to
Since the invention of the rst light bulb by Thomas Alva Edison reveal the invisible has made them one of the most important
in 1879, light sources have been the primary tools for the investi- research and diagnostic tools in medicine, chemistry, and physics in
gation of matter. But it was the discovery of X-rays in 1895 by the last century. One of the most powerful sources of X-rays in
modern research are synchrotrons, in which electrons are acceler-
ated by radiofrequency cavities to extremely high energies
Abbreviations: XFEL, X-ray free electron laser; FEL, free electron laser; SFX, serial (typically  3 GeV) and transferred to a storage ring in which they
femtosecond crystallography; DESY, Deutsches Elektronen-Synchrotron; LCLS, Linac can be maintained at high current in stable bunches for hours. The
Coherent Light Source; SACLA, Spring-8 Angstrom Compact free electron LAser; PSI, electrons are then subsequently perturbed in an undulating elec-
photosystem I; DLS, dynamic light scattering; SONICC, second order of non-linear
tromagnetic eld to induce X-ray emission. The rst synchrotron
imaging of chiral crystals; SHG, second harmonic generation; CSPAD, Cornell
SLAC pixel array detector; FID, free interface diffusion; LCP, lipidic cubic phase; facility was built in the early 1970's and due to the increasing de-
TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis; GDVN, mand from both the scientic community and private research
gas dynamic virtual nozzle; HPLC, high performance liquid chromatography; TR- sector; the number of synchrotron facilities has grown quickly
SFX, time-resolved serial femtosecond crystallography; CCD, charged coupled de- since. In the last three decades, synchrotrons have been built or are
vice; HI-RIP, high-intensity radiation induced phasing; MIR, multiple isomorphous
in the construction phase in 24 countries. APS at the Argonne Na-
replacement; MAD, multi-wavelength anomalous dispersion; SAD, single-wave-
length anomalous dispersion. tional Laboratory (Chicago, USA), the ESRF (Grenoble, France),
This article is part of a Special Issue entitled Protein Crystallography, edited by PETRA III (Hamburg, Germany), Spring-8 (Harima Science Park City,
Ana Camara-Artigas and Jose Antonio Gavira. Japan), and DIAMOND (Oxfordshire, England) are currently the
* Corresponding author. School of Molecular Sciences, Arizona State University, largest and most powerful synchrotron light sources. X-ray
Tempe, AZ 85287-1604, USA.
E-mail address: (P. Fromme).
methods have also played an increasingly more important role in
0003-9861/Published by Elsevier Inc.
J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47 33

the life sciences as evidenced by medical X-ray imaging, X-ray causes a massive increase in ux compared to synchrotron sources.
scattering, and highlighted here, X-ray crystallography. The latter Furthermore, the high longitudinal coherence (and thus, ultrashort
typically makes use of X-rays from 3.5 keV to 20 keV (3.5 to 0.6 ) duration) of the pulses pushes the peak brilliance orders of
to enable the determination of the atomic structure of matter. The magnitude higher than that achievable at a synchrotron. The
most difcult and challenging biomolecules, such as large com- output wavelength can by tuned by modulating the electron energy
plexes and membrane proteins, are almost exclusively solved with and magnetic eld strength. Fig. 1 illustrates this principle. XFELs
synchrotron radiation. However, in spite of the usefulness and generate high gain, ultrashort pulsed X-rays with only a single
power of these facilities, there are two important areas in which undulator pass [6], accomplished by the interaction between
synchrotron facilities are limited in addressing the full range of oscillating electrons, moving at relativistic speeds, with their
current scientic challenges. The main limitations are primary and emitted electromagnetic waves (for a more detailed overview of
secondary X-ray damage which cannot be outrun by pulse dura- XFEL physics please refer to Ackermann et al. [7] and McNeil et al.
tions currently available at synchrotron sources (tens of ps). Sec- [8]). The interaction between the electrons and their radiation
ondary damage can be minimized by freezing but primary damage causes spatial redistribution of the electrons into compact bunches
is unavoidable. perpendicular to their direction of motion. This generates coherent
To overcome these limitations a new light source has emerged X-ray pulses with durations of tens of femtoseconds. The rst hard
in the form of the X-ray free electron laser (XFEL) which has XFEL, LCLS, was built at the SLAC National Accelerator Laboratory in
improved upon many of the properties of synchrotron radiation California and has been in operation since April 2009 [9]. It pro-
sources, in some cases by orders of magnitude. Free electron lasers duces X-ray pulses of approximately 3 mJ energy at 120 Hz. Each
(FELs) have been used over many years since their conception by pulse has a duration that ranges from around 300 fs down to a few
John Madey in the early 70's [1], initially operating at infrared femtoseconds with up to 1013 coherent photons per pulse. Thus, the
wavelengths. More recently, visible and near ultraviolet wave- LCLS instrument has set a new standard, with a peak X-ray bril-
lengths were achieved [2]. Since its discovery, the idea of extending liance over ten orders of magnitude higher than that of the most
FELs to shorter wavelengths, in particular to the X-ray regime, have powerful synchrotron radiation sources [10]. XFELs are unique light
been considered and explored by many scientists (see Ref. [2] for a sources that can be used to explore matter at atomic length and
review on the development of FELs). After many years of theoretical femtosecond time scales. The increase in brightness along with
and experimental work along with new technological advances, ultra-short pulses has facilitated the appearance of a new applica-
three XFELs are currently operational. Namely, they are the tion of XFEL technology in the eld of structural biology via serial
Deutsches Elektronen-Synchrotron's (DESY) Free-electron LASer in femtosecond crystallography (SFX) [11,12]. A typical setup for SFX
Hamburg (FLASH), SLAC's Linac Coherent Light Source (LCLS), and data collection is illustrated in Fig. 2.
RIKEN's Spring-8 Angstrom Compact free electron LAser (SACLA). The technique of macromolecular X-ray crystallography and its
At its core, an XFEL consists of an electron source, a linear accel- historical success in determining the structure of biological mac-
erator, and undulator magnets spaced to produce X-ray wave- romolecules has always suffered from a major bottleneck, namely
lengths (typically ranging from 0.01 to 10 nm) [1,3,4]. Briey, a the production of well diffracting crystals. This problem is espe-
relativistic electron beam is accelerated to almost the speed of light cially prevalent in the structure determination of membrane pro-
in a linear accelerator prior to interaction with the undulator. Upon teins, which are notorious for their difculty in forming high
interaction with the undulator, the electrons move in curved paths diffraction quality crystals. In some cases, years have been devoted
via the magnets. The long length of an XFEL undulator allows the to determining crystallization conditions for a membrane protein
relativistic electrons to interact with their emitted radiation which and it is not uncommon that only showers of nanocrystals (crystals
causes bunching of the electrons with spacing equal to the wave- between 200 nm and 10 mm) are observed while attempts to grow
length of the emitted X-rays. As the electrons bunch, their emission larger crystals remain unsuccessful. These nanocrystals were once
becomes more coherent and allows for a stronger interaction be- seen as only a possible intermediate towards achieving useable
tween the two. This results in beam with highly coherent pulses crystals [13]. Modern microfocus beamlines have expanded the
with femtosecond duration. The coherence causes radiative emis- usefulness of these nanocrystals but even they experience con-
sion proportional to the number of electrons squared in contrast to straints on what is achievable mainly due to severe X-ray damage
a synchrotron (incoherent) in which the radiative emission scales by long exposure times, often requiring cryo-cooling and necessi-
proportional to the number of electrons due to cancelation effects tating crystals larger than 10 mm, which must be even larger in the
from the out of phase generated electromagnetic elds [5]. Because case of large unit cells (common in membrane proteins and large
the number of electrons in a bunch is on the order of a billion, this complexes) [14]. With the advent of XFELs, crystals which would

Fig. 1. Schematic representation of an undulator segment at an XFEL instrument. A relativistic electron beam (solid line) is brought to high energy in a linear accelerator (not
pictured) prior to interaction with the undulator. The electrons then travel on a sinusoidal path, induced by a special arrangement of magnets called an undulator, a periodic array of
magnetic dipoles shown as red and blue boxes. Because the electrons move in curved paths via the magnets, the change in momentum causes the emission of monochromatic
radiation the same way a synchrotron does (depicted as a red cone).
34 J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47

Fig. 2. Schematic representation of the experimental setup of a typical SFX experiment at LCLS. Randomly oriented nanocrystals (green) in their mother liquor are delivered into the
focus of X-ray beam by a gas-focused liquid injector. The X-ray beam, which is transverse to the jet, hits the crystals in the interaction region. Diffraction snapshots of single crystals
are recorded using a Cornell-SLAC pixel array detector (CSPAD) located in the forward-scattering region.

have previously been too small for use have been shown to be the native oxidation state. This effect was still seen at 4% of average
suitable for structure determination [11]. Generally, these small doses used for crystallography [28].
crystals are easier to produce and possess signicantly less long In 2000 Neutze et al. [29] conducted molecular dynamic simu-
range disorder. Furthermore, nanocrystals are ideal for time- lations on the time course of the Coulomb explosion of T4 lysozyme
resolved studies as a greater percentage of molecules in the crys- at a total ux of 3.8  106 12 keV photons per 2 over pulse dura-
tal can be activated homogeneously by light or rapid mixing. For tions of 2, 10, and 50 fs (the presumed theoretical ux achievable by
light activated reactions, time domains ranging from the femto- an XFEL) in vacuum. At this time, XFELs were still in the planning
second to microsecond regimes can be probed and temporal res- phases. They predicted that the onset of the Coulomb explosion
olution of reactions induced by mixing can range from seconds would occur at 5e10 fs and predicted that if the pulses are short,
down to microseconds [15e19]. diffraction of the molecule could be collected before it is destroyed
In addition to the challenges of sample preparation, the problem referred to as diffraction before destruction. This principle relies
of X-ray induced radiation damage has hindered progress in on an XFELs ability to deliver a sufcient number of photons (~1013
structure determination even in well-diffracting crystals. Tradi- photons/pulse, orders of magnitude greater than that required to
tionally, cryogenic cooling of crystals has been the most successful form a plasma) for structure determination on time scales
way to minimize damage, increasing the radiation dose tolerance competing with primary radiation damage events. The rst
by a factor between 30 and 50 [2022]. Cryo-cooling works by experimental demonstration of this principle was carried out using
slowing the dispersion of radiation-induced reactive products that the soft XFEL, FLASH, located at DESY [30]. In this experiment an
occur during the X-ray exposure. Upon exposure to X-rays, photo- intense 25 fs, 4  103 W/cm2 pulse, containing 1012 photons, pro-
ionization and processes related to Auger decay take place, leading duced a coherent diffraction pattern from a non-periodic object
to heat as well as the production of radicals and photo-ions. These before destruction occurred [31]. Thus, XFELs have the potential to
diffuse within the crystal at rates dependent on available kinetic minimize the effects of radiation damage and reduce the size re-
energy. This can lead to subsequent chemical reactions that result strictions on crystals suitable for X-ray structure determination.
in the breaking of chemical bonds and thus a reduction in the SFX requires new data processing and handling as the determina-
diffraction quality of the crystal. While cryo-cooling does not in- tion of crystallographic structures is based on thousands of snap-
uence the primary X-ray damage (i.e. photoionization and Auger shot diffraction patterns at room temperature by continuous
decay), it slows down diffusion of the secondary processes of X-ray delivery of orientationally unrelated nanocrystals in their mother
induced damage, radicals and heat progression. This method thus liquor [11,32e36].
allows crystals to tolerate longer exposure times, improving the In December 2009 the rst SFX experiment was carried out at
signal to noise ratio. Nonetheless, even at cryogenic temperatures LCLS, using nanocrystals of photosystem I (PSI) as the rst sample.
site-specic radiation damage remains a problem, in which rapid PSI mediates the conversion of light energy from the sun to
photo-damage by the X-ray beam in areas with high X-ray cross- chemical energy in plants, green algae, and cyanobacteria, is one of
sections can occur, decreasing diffraction until it ultimately termi- the most complex membrane proteins crystalized so far, the entire
nates. In such cases, partial data sets from many crystals must be complex consisting of 36 proteins and 381 cofactors [37]. Tens of
merged in order to obtain a structural model. Furthermore, these thousands of diffraction patterns were collected, which allowed for
models can exhibit signicant artifacts from damage which can be the determination of the PSI structure, providing a proof of concept
substantially different from the native structure [23]. Examples of for SFX [11]. Furthermore, this study resolved interference fringes
radiation damage include the breakage of disulde bonds and salt between the Bragg peaks which were originally suggested by Sayre
bridges, tyrosine residues becoming hydrolyzed, decarboxylation of in 1952 [38]. He proposed that diffraction from crystals with a
glutamate and aspartate residues, B-factor increase, and loss of countable number of unit cells, would show the Fourier transforms
diffraction progressing from high to low resolution [24e27]. Radi- of the crystals in the diffraction pattern leading to fringes between
ation damage is particularly troublesome in limiting resolution of the Bragg peaks directly related to the number of unit cells (n1)
high-Z catalytic centers, causing metalloproteins to be the most [39]. This theory came to fruition in the rst SFX experiments when
prone to local radiation damage. For example, spectroscopy has these fringes were detected in the diffraction patterns. In the future,
shown that X-ray radiation exposure to cryo-cooled, photosystem II with higher spatial resolution detectors, these shape transforms
crystals leads to the metal center becoming photo-reduced below can be used for direct phasing [40].
J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47 35

Six years after the rst SFX experiments were carried out [11], of a proteins phase space is highly benecial for the optimization of
evidence has continued to mount that XFELs make overcoming crystallization conditions. Driven by thermodynamics, the forma-
radiation damage in protein crystallography an attainable reali- tion of protein crystals requires a controlled decrease in solubility
zation [27,32,33,41e52]. In addition, SFX not only mitigates radi- of the protein that ideally avoids the formation of non-ordered
ation damage but is suitable for crystals with as little as a few precipitation. Traditionally, the supersaturated area of phase
hundred unit cells. Thereby, data can be collected from nano- space is typically reached by gradually increasing the precipitant
crystals eliminating the need to grow large crystals. Experimental concentration, allowing for few nucleation sites to form that grow
evidence is emerging that nanocrystals may show signicantly less by addition of the free protein to the nuclei over time, resulting in a
long-range disorder than their larger counterparts, making them few large crystals [56]. In contrast to this approach, nano-
ideal candidates for the structure determination of challenging crystallization is best achieved by inducing a high number of
proteins. nucleation sites, whereby the free protein concentration is rapidly
decreased by the formation of a plethora of nuclei followed by their
2. Nanocrystallization and characterization growth into a multitude of small crystals [54,57]. Typically this is
achieved by increase of the concentration of protein, precipitating
Because nanocrystals were previously seen as merely a stepping agents, or both as compared to optimized conditions for large
stone for the desired growth of large crystals, nanocrystal growth crystal growth for the same protein [54]. While the approaches for
methods have remained largely unexplored. Due to the serial na- each size regime differ, they both rely fundamentally on the
ture of SFX, a few unique characteristics must be considered for the knowledge of the proteins phase space and may be even more
development of nanocrystallization techniques. First, a different important for growth of well-ordered nanocrystals as formation of
crystal in a random orientation is used for each diffraction pattern, amorphous precipitate is best avoided.
with patterns being captured in a serial fashion [11]. Thus, to Many of the existing methods for macro-crystallogenesis are
constantly replenish the sample between X-ray pulses, crystals are also applicable for nanocrystallization with modication necessary
delivered to the X-ray interaction region by a liquid jet (in a typical to occupy a different area of phase space [45,54,58e61]. Batch
SFX experiment) composed of crystals in their mother liquor at methods, in which the protein and precipitating conditions are
room temperature [11]. The delivery of the crystals to the X-ray mixed to homogeneity initially, provide a good example of well-
region is much more rapid than the X-ray repetition rate. Conse- established methodology that needs little modication for suit-
quently, most crystals do not interact with the X-rays [35]. Addi- ability in SFX (outside of parameter values). This is illustrated in
tionally, since diffraction patterns represent still frame slices Fig. 3 where a batch approach for macro- and nanocrystals differs
through the Ewald sphere, only partial reections can be recorded only by starting point within the phase space [62]. It has been re-
(i.e. there is no goniometer and therefore no rotation to record full ported in some cases that large crystals grown by traditional
proles of reections). In order to measure accurate structure fac- methods can also be mechanically crushed to obtain the smaller
tors of the Miller indices (h, k, l), high redundancy of the data sets crystals needed for SFX [51,63]. This is, however, not generally
(>50) are required via Monte Carlo data analysis methods [53]. Due applicable and may lead to loss of quality or destruction of fragile
to approximately one in every ten thousand crystals resulting in a crystals, such as those common amongst membrane proteins.
diffraction pattern and the necessity for high multiplicity of the Nanocrystalline showers with traditional methods such as vapor
data, tens to hundreds of milligrams of sample may be needed for diffusion have been obtained, using a brute force approach with
successful SFX data sets, where samples are delivered in a liquid jet. many individual setups to scale for SFX experiments [64]. While
The hit rate (percentage of pulses that result in crystal diffraction) is this poses no theoretical issue, the concern for this type of nano-
largely a function of crystal density and as such, must be considered crystal preparation is due to the tedious nature of sample prepa-
in growth methods and characterization of the crystals. ration involved to obtain enough material for full data set
Since data from many crystals must be merged in analysis of SFX collection. While this can in part be alleviated with sample
data, it is also important to consider crystal homogeneity. For conserving delivery systems [61,65,66] (assuming they are
example, if there is a large size distribution in the sample, then the compatible), sample density and homogeneity remain challenging
data set will be composed of diffraction patterns with varying and necessitate specic consideration.
relative Bragg peak intensities (directly related to the number of The nanocrystalline free interface diffusion (FID) method is an
unit cells and hence, the size). This can lead to scaling issues which example of a technique that has been developed from an existing
become problematic in subsequent analysis. Furthermore, the method for SFX applications [54]. In this method, one solution
presence of some outlying (e.g. larger) crystals may necessitate (either the protein or precipitant solution) is added drop wise to the
attenuation of the beam to prevent damage to the detector. This in other. The less dense of the two (typically the protein/buffer solu-
turn would limit resolution on images obtained from the more tion) is rst placed in a vessel, typically a microcentrifuge tube. The
plentiful smaller crystals. Size inhomogeneity can also lead to in- denser solution (typically precipitants such as high concentration
stabilities or clogging in the liquid jet [54]. Therefore, it is imper- salts, polyethylene glycol, etc.) is then added drop wise, thereby
ative to consider and monitor crystal size distribution while allowing for a high nucleation rate at the protein/precipitant
screening conditions for nanocrystallization. These considerations interface of the two solutions but still inducing some mixing
become even more pronounced in time-resolved experiments immediately. Nanocrystals will form at the interface, owing to the
where the crystal size is the primary parameter in reaction initia- access of areas of phase space that typically results in high nucle-
tion homogeneity [16,55]. To meet the need for large sample vol- ation rates (i.e. high concentrations of both protein and pre-
umes, optimized crystal density and crystal size homogeneity, in cipitants). Additional benets arise in the common scenario where
addition to the traditional optimization of crystal diffraction qual- the denser solution is the precipitant since this allows crystals that
ity, nanocrystallization techniques are rapidly emerging to address form at the interface to settle due to gravity into the precipitant rich
the unique challenges present in SFX. layer. This provides a way to effectively quench crystal growth as
well as accumulate a high density of nanocrystals, allowing prac-
2.1. Nanocrystallogenesis tical optimization of crystal density by resuspension to a desired
concentration. Gentle centrifugation of the setup has also been
As is the case in the growth of macroscopic crystals, knowledge shown to expedite crystal formation (e.g. photosystem II forming
36 J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47

Fig. 3. A) 2-dimensional slice of a typical free protein phase diagram with selected crystallogenesis methods exemplifying the general relationship between phase space occupation
and resultant crystalline protein. B) Depiction of the microcrystalline FID method in the case of a denser precipitant being dropped through a protein solution and the subsequent
interface resulting in microcrystal pelleting (B reproduced with permission from Ref. [17]).

nanocrystals in as little as 30 min with centrifugation in contrast to harvesting the crystal pellet or removal of the free protein layer
1 day without centrifugation [54]) and has led to improvement of before considerable mixing occurs.
crystal size and homogeneity due to the tendency for crystals to
spend less time in relatively high concentrations of free protein 2.2. In-vivo crystallization
after nuclei formation, resulting in more uniform growth
throughout the sample. It is important to note that over time, full Crystallization in vivo using insect and mammalian cells is a
mixing of the two layers will occur (rate proportional to misci- highly innovative approach towards nanocrystallography that has
bility). This could lead to the dissolution of the crystals if the been recently discovered [49,51,67,68]. This technique was rst
mixture represents an undersaturated portion of phase space. applied in SFX in 2012 after nanocrystals were identied by elec-
Oswald ripening and decreased size homogeneity can also occur if tron microscopy inside insect cells when cathepsin B from Trypa-
there is high mobility or an appreciable amount of free protein left nosoma brucei was overexpressed using the recombinant
over after complete mixing has occurred. This can be avoided by baculovirus system [49]. Redecke and co-workers observed needle
J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47 37

shaped nanocrystals protruding out of the cell after 70 h but were [77]. SONICC can identify nanocrystals of chiral molecules as small
still surrounded by the cell membrane [51]. The crystals were as 100 nm [77]. When a chiral crystal is exposed to two 1024 nm
10e15 mm in length and about 0.5e1 mm in width. These crystals photons in a strong eld, frequency doubling occurs due to inherent
were isolated and used for SFX analysis [51]. It has been proposed polarization anisotropy, allowing a detector to measure the 512 nm
that this spontaneous crystallization may occur due to accumula- photon output. Constructive interference arising from crystalline
tion of the protein in a specic organelle such as the endoplasmic translational symmetry increases the probability of this occurring,
reticulum, peroxisomes, or secretory granules [49,69]. In vivo allowing for practical measurements to be taken. It should be
crystallization is not limited to the Sf9 insect cells and has been cautioned that the signal strength depends on the space group
demonstrated for multiple proteins in cockroaches, seeds, and (with higher symmetry leading to decreased signal in general) and
bacterial cells [52,67,69]. However, little is understood about the also the specic molecular polarization susceptibility (higher SHG
mechanism of in-vivo crystallization as it was initially hypothesized is typical in molecules with a chromophore) [78]. While truly
to be a rare occurrence. Additionally, the crystals were not probed centrosymmetric space groups are impossible in natural protein
when they were rst found as they were too small for macro- crystals, many of the high symmetry space groups can still lead to
crystallography [49,69]. With the advent of XFELs, in vivo crystal- attenuation of signal below detection, resulting in false negatives.
lization could potentially become a major method for crystalliza- Furthermore, some precipitants are chiral (e.g. sugars, chiral salts)
tion, thus removing the bottleneck of determining crystallization and can crystallize in space groups that can be active in SHG (i.e.
conditions for applicable proteins. Before in vivo crystallization false positives) so care must be taken when interpreting results.
comes to fruition as a more general method, further research needs The most trustworthy method to verify the existence of dif-
to be done towards understanding the necessary mechanistic fracting crystals is X-ray powder diffraction, which can be carried
components involved. out at either cryogenic or ambient temperatures. For room tem-
perature powder diffraction measurements, a high density pellet of
2.3. Growing crystals using LCP crystals is transferred to an X-ray transparent capillary [79e81]. In
contrast with cryogenic powder diffraction, room temperature
Lipidic cubic phase (LCP), a bicontinuous mesophase that acts as powder diffraction measurements are ideal for optimizing relative
a membrane-mimetic, has been used to crystallize a variety of resolution for an XFEL since this ensures that there are not artifacts
membrane proteins, notably G coupled-protein receptors, ion arising from the freezing process. It is also important to include
channels, and transporters [70,71]. Unlike membrane protein buffer in the capillary to avoid drying out of the small crystals
crystals grown in surfo (in partial or full detergent micelles), which which can affect diffraction quality. While powder diffraction data
usually exhibit type II micelle crystal packing and often have a high can be collected from nanocrystals at low ux X-ray home sources,
solvent content, crystals grown in LCP feature type I crystal packing it requires several microliters of dense crystal sample which is more
[72]. This packing allows for hydrophilic protein-protein and hy- than is produced from commercial screens [63]. It also depends
drophobic protein-lipid-protein interactions and thus, often leads highly on the sample quality and density so establishing conditions
to tighter and more rigid packing, in turn possibly leading to a for powder diffraction typically requires synchrotron radiation to
lower solvent content and better diffraction [73]. To crystallize produce measurable diffraction for un-optimized samples [82].
membrane proteins in LCP, puried protein at high concentration Transmission electron microscopy (TEM) is another reliable
(usually > 20 mg/mL) is mixed with molten monoolein in Hamilton method to monitor nanocrystal quality and requires low sample
gas-tight syringes using a syringe-mixer [74]. For standard crys- volumes. Strong correlations exist between crystals that have
tallography, special robots (e.g. Flexus Crystal IMP, Gryphon LCP, highly ordered lattices visible through TEM and crystals that
NT8-LCP, Mosquito LCP, ProCrys Meso) can be used to dispense the feature better X-ray diffraction quality [82]. While this technique
LCP and precipitant and crystals are often grown in micro-batch can provide insightful characterization, it requires elaborate sample
assays of <1 nLe100 nL. Cherezov and colleagues have had preparation and often involves negative staining which can affect
notable success in adapting the LCP crystallization method for SFX electron diffraction and is only suitable for very thin nanocrystals
[27,44,73,75,76]. In this method, protein laden LCP is injected in (<200 nm) [82,83]. Large crystals can be mechanically crushed in
another syringe containing precipitate solution and incubated for order to achieve dimensions suitable for TEM [63].
24 h to two weeks to permit crystallization [27,75]. Excess precip- In protein crystallography, dynamic light scattering (DLS) is
itate solution is removed and the crystals embedded in the LCP are commonly used to analyze protein homogeneity since a mono-
delivered using a high-viscosity-injector [35]. disperse (i.e. homogenous and non-aggregated) sample is ideal for
crystallization [84]. In addition, DLS is also used in SFX experiments
2.4. Crystal detection and characterization to determine nanocrystal size distribution and homogeneity across
multiple conditions [85]. DLS measures the light scattered by par-
Some SFX experiments have been carried out with crystals that ticles in solution. As molecules in solution undergo Brownian mo-
are >5 mm and can therefore be identied by established methods tion, the change in intermolecular distance leads to constructive
such as polarized light microscopy in combination with UV- and destructive interference of the scattered light. Fluctuations in
uorescence microscopy. Nanocrystals are very difcult to iden- intensity over time are indicative of the particle size and can be
tify and differentiate from amorphous precipitate, particularly if the derived from the Stokes-Einstein relation. DLS only requires a few
crystal size is on the order or smaller than 1 mm, nearing the limit of microliters of sample for analysis at broad range of concentrations
resolution for optical microscopy methods. Techniques such as (about 108e1012 crystals/mL) [86]. However, large particles such as
tryptophan uorescence and birefringence may also be limited dust or aggregates can affect the accuracy of size determination
since the signal for each is proportional to crystal size. Alternative [87]. Another important parameter to take into account in SFX
methods for detecting small crystals have proven to overcome the experiments is the crystal density. Crystal density can be optimized
aforementioned difculties. One of the most useful methods for by using nanoparticle tracking analysis (NTA), which, like DLS, in-
rapid feedback during initial and optimization stages of crystallo- fers the Brownian motion of particles in solution and relates this
genesis is the use of second-order harmonic generation (SHG) movement to an equivalent hydrodynamic radius. Unlike DLS, NTA
spectroscopy, in particular the SONICC (second order non-linear uses video to track the Brownian motion and thus, captures the
imaging of chiral crystals) instrument invented by G. Simpson light scattering signal from individual particles [88]. This not only
38 J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47

allows for particle size to be determined on a particle-by-particle has shown great success and breadth, limitations of this method
basis but also allows for particle concentration to be estimated have trigged multiple strategies to deliver crystals to the X-ray
which is extremely helpful for optimizing crystal density for SFX interaction region to be born over the last 6 years. In this section the
experiments [86]. However only particles <1 mm can be detected current crystal delivery methods used at XFELs are reviewed
and 1 mL of particle concentrations of 107e109 crystals/mL [86] is (summarized in Table 1).
required. Therefore, DLS and NTA provide a means to monitor
nanocrystal size. Furthermore, microuidic devices using dielec-
trophoresis have been invented that can sort nanocrystals accord- 3.1. Gas-focused liquid injectors
ing to size [89].
In preparation for the rst SFX experiments, the gas dynamic
virtual nozzle (GDVN) was invented to continuously deliver crystals
3. Sample delivery methods to the X-ray pulses [36,90]. Based on the principle that owing gas
encompassing a liquid can focus the liquid jet to a smaller diameter
The majority of SFX data has been collected from a jet of small (1/10 the original capillary size) the GDVN produces a liquid jet that
crystals (typically 200 nme10 mm) in their mother liquor. This is only a few microns in diameter [36,90]. This is achieved by
scheme allows the sample to be constantly replenished for each mounting a smaller capillary inside a larger capillary. Crystals in
XFEL pulse as the crystals are destroyed with each shot. While this their mother liquor are delivered through the smaller, inner

Table 1
Summary of sample delivery methods currently being deployed for SFX.

Sample delivery Average Advantages Limitations Experimental Examples of systems/Proteins studied

method ow rate environment

Gas dynamic 10-25 mL/ Amenable for TR-SFX [17,41], compatible High sample consumption [34,35,61], Vacuum Photosystem I & II [17,98],
virtual nozzle min [62] with most systems freezing around nozzle tip [34], fragile photosystem I-ferredoxin [41],
[90] crystals might be damaged during lysozyme [32], cathepsin B [51],
jetting [82,91], requires crystals to be photosynthetic reaction center
ltered [107] [46],Cry3A [52], photoactive yellow
protein [16], P-type ATPase [58],
myoglobin [18], phycocyanin [66], B
subunit of cholera toxin-membrane-
proximal region of gp41 [99], CPV17
polyhedrin [68], trans-acting acyl
transferase [63], RNA polymerase B II
[63], parathyroid hormone receptor 1
Electrospinning 140 Low sample consumption [33], Requires cryoprotectant to prevent the Vacuum Photosystem II [100], 30S ribosomal
[33] e3100 nL/ compatible with membrane proteins jet from freezing [33], unknown if high subunit [91], thermolysin [101]
min [33] [100] electric elds affect crystals [65]
Lipidic cubic 50 Low sample consumption [27], crystals Debye Sherrer rings [65], membrane Both 5-HT2B receptor [27], smoothened
phase [35] e200 nL/ grown in LCP do not need to be harvested protein crystals cannot be mixed post receptor [35], human delta opioid
min [27] [27], some soluble crystals post- crystallization [65], crystals grown in receptor [44], human angiotensin
crystallization can be mixed to conserve LCP cannot be ltered before injection receptor [76], rhodopsin arrestin
sample [66] complex [48], diacylglycerol kinase
[102], lysozyme [66], phycocyanin [66]
Grease [61] 460 Low sample consumption [61] Debye-Sherrer rings [65], not shown to Atmosphere Lysozyme [61], glucose isomerase [61],
e480 nL/ be compatible with membrane proteins thaumatin [61], fatty acidebinding
min [61] [65] protein type 3 [61], luciferin-
regenerating enzyme [60]
Agarose [65] 100 Low sample consumption [65], Requires cryoprotectant to prevent the Both Phycocyanin [65], photosystem I & II
e200 nL/ compatible with membrane and soluble jet from freezing when operated in [65], sindbis virus [64]
min [65] proteins [65], low background [65] vacuum [65]
Fixed target: NA Low sample consumption [105], Commercial wafers/chips material is Both Rapid encystment protein 24 kDa [45],
sample compatible with 2D crystals [103], target not X-ray transparent [104,105], low bacteriorhodopsin [103], anthrax toxin
supports can be rotated about a xed angle [105] data collection rates (<10 hz, limited by protective antigen [104], lysozyme
(wafers velocity of sample holder) [45], crystal [105], sperm whale myoglobin [106]
[45,103,104] traps can produce crystal orientation
and chips biases [105] and ll rate is limited to
[105,106]) ~50% to prevent crystal stacking [106],
in vacuum experiments requires
emerging crystals in oil [45,104], unless
crystals are grown on a chip they must
be transferred [105,106]
Fixed target: NA Amenable to micro- and to macro- crystals Requires cryopreservation and freezing Atmosphere RNA polymerase II [107], general
goniometer [107], compatible with standard [107], not amenable for TR-SFX, transcription factor IIB [107], large
[107] (Grids/ goniometers [107] requires hitting the crystal in a different nucleic acid scaffold [107], monomeric
Mesh/Loops) position each shot to avoid damage iron-containing hydrogenase [107],
sperm whale myoglobin [107],
cytochrome c oxidase [108],
synaptotagmin-1 SNARE complex
[109], b2-adrenoreceptor/nanobody
complex [107], cyclophilin A [110]
J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47 39

capillary and high-pressured gas ows through the interstitial space liquid chromatography (HPLC) valve, as to minimize sample loss
between the larger, outer capillary and the inner capillary (see between pulses. It has been reported that ow times of 300 ms
Fig. 4). The GDVN has been used successfully for many SFX experi- separated by times of 2 ms without ow could reduce the GDVN
ments (see Table 1) including time-resolved studies [17,41,51] and sample consumption by a factor of four [34]. Similar to a pulsed
has thus far been the workhorse for the majority of SFX experi- method, droplets can be used to deliver crystals to the X-ray beam.
ments. While the GDVN has experienced much success, several In acoustic droplet ejection, droplets can be produced by a piezo-
limitations exist. Namely, clogging of the smaller, inner capillary can electric transducer [94]. To optimally synchronize the crystal lled
occur due to blockage from aggregating crystals, the shear forces drops, the droplets would be trigged by the XFEL. However, droplet
experienced during jetting have been suspected to damage some speeds produced tend to be unstable on the necessary timescales,
fragile crystals [82,91], and ice can form on the nozzle by back- making synchronization difcult. Additional challenges faced by
spraying of debris from the explosion at the X-ray interaction re- this method include large droplet size and thus high background
gion [90,91]. Clogging events can be decreased by ltering the [95,96], crystal settling without the addition of a viscous solution
sample prior to sample loading and using in-line lters upstream [34], and compatibility with high vacuum [34]. To date, neither the
from the nozzle. However, ltering could result in loss of sample and pulsed liquid jet nor acoustic droplet ejection has been employed at
damage to crystals, thus highlighting the need for knowledge and an XFEL successfully.
control over the size distribution [91]. One challenge when using the
GDVN is that the X-ray pulse repetition rates currently available 3.2. Electrospinning injectors
(120 Hz) are low compared to the minimal ow rate of the GDVN at
10 mL/min, thus the majority of crystals never interact with the X-ray In one method of counteracting the fast consumption of sample
beam since they ow through the interaction region between pulses by the GDVN, an electrospun liquid microjet, which uses high
[34]. Therefore, large sample volumes are needed to produce a electric elds instead of gas to focus the liquid, has been imple-
complete SFX dataset using this method. Data sets using the GDVN mented [33]. Electrospinning allows for a slower moving jet and
can require 10 mL of crystal suspensions, typically containing on the thus, fewer sample ows between XFEL pulses and the amount of
order of 109e1011 crystals/mL [11,54,92,93]. (see Table 2). sample needed for a dataset can be reduced [35]. The slow speed is
Several strategies have been proposed to reduce the ow rate of also suitable for experiments with long pump-probe delay times as
the GDVN. First, the GDVN can be operated in a pulsed mode it allows for longer incubation times compared to those achievable
whereby the jet is switched on and off, using a high performance with the GDVN [33]. However, to form a continuous jet and prevent
crystal settling, the crystals must be suspended in a suitable viscous
media (e.g. glycerol, PEG, or sucrose); otherwise the electrospun jet
breaks up into highly charged droplets once surface tension is
overcome. The embedding media also serves as a cryo-protectant,
necessary to prevent jet dehydration due to the length of time
spent in vacuum, making this method not universally suitable.
Caution of the impact of the electric eld and the high charge of the
jet and droplets on the sample must also be considered as they are
unknown at present [33].

3.3. High-viscosity media injectors

LCP has a viscosity similar to vacuum grease, which makes

harvesting tens of thousands of small crystals from their sticky,
viscous environment impractical. Thus, an alternative method was
devised so that the crystal laden LCP could be delivered directly to
an X-ray beam. To extrude such a viscous material, the high-
viscosity injector (commonly referred to as the LCP injector)
amplies the pressure of an HPLC using a hydraulic stage to extrude
the viscous media out of a capillary. A co-owing gas stream en-
sures that the highly viscous uid does not curl back onto the
nozzle and forms a stable jet [35]. Unlike the GDVN, the jet is not
focused to a smaller size and maintains the inner diameter of the
capillary it is extruded from (10e100 mm). In addition, the high
viscosity injector has been shown to work both in vacuum and in air
[97]. To decrease sample consumption for soluble nanocrystals,
crystals have been successfully manually mixed post-crystallization
with LCP and grease [61,66]. Recently, agarose-based gels have been
shown to be compatible for the sample delivery of both soluble and
membrane proteins which are embedded into pre-gelled agarose
using a coupled syringe setup as described in Conrad et al. [65]. The
agarose jet also offers an improved background over grease [61] or
LCP, leading to higher quality data [65].
Fig. 4. Schematic of GDVN. The GDVN is assembled by placing a smaller, hollow-core
fused silica optical ber (inner capillary) inside a larger, borosilicate glass capillary 3.4. Fixed targets
(outer capillary). Crystals are passed through the inner capillary while a focusing gas is
passed through the outer capillary and thus occupies the space in between the two
capillaries. A thin, micrometer jet is produced when the co-owing gas meets the For most experiments that use a owing jet, either liquid,
crystals as they exit the inner capillary. The scale bar is 0.1 mm. viscous, or electrospun, the optimal crystal hit rate is usually
40 J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47

Table 2
Details of the structures depicted in Fig. 5.

Protein name Organism PDB Protein Resolution Space Unit cell Rwork, Rfree Year Reference
code classication group

Photosystem I Thermosynechococcus 3PCQ Membrane 8.98 P63 281.0, 281.0, 165.2 0.25, 0.23 2010a (Chapman et al.,
elongatus 90, 90, 120 2011)
FABP3 Homo sapiens 3WXQ Soluble 1.6 P212121 133.25, 226.26, 0.178, 2014b (Sugahara et al.,
307.09 0.222 2015)
90, 90, 90
Purple bacterial reaction Blastochloris viridis 4CAS Membrane 3.5 P212121 104.8, 104.8, 104.8 0.295, 2013a (Johansson et al.,
center 90, 90, 90 0.329 2013)
Lysozyme Gallus gallus 4ET8 Soluble 1.9 P43212 33.71, 54.85, 70,66 0.196, 2012a (Boutet et al., 2012)
90, 90, 90 0.229
Cathepsin B Trypanosoma brucei 4HWY Soluble 2.1 P42212 57.90, 84.80, 0.182, 2012a (Redecke et al.,
384.30 0.213 2013)
90, 90, 90
Serotonin receptor 5-HT2B Homo sapiens 4NC3 Membrane 2.8 C2221 79.0, 79.0, 38.0 0.227, 2013a (Liu et al., 2013)
90, 90, 90 0.270
Photosystem II Thermosynechococcus 4PBU Membrane 5.0 P212121 125.40, 125.40, 0.262, 2014a (Kupitz et al., 2014)
elongatus 54.56 0.261
90, 90, 90
-opioid receptor
a Homo sapiens 4RWD Membrane 2.7 C121 61.50, 122.20, 0.208, 2014a (Fenalti et al., 2015)
168.50 0.238
90, 90, 90
smoothened receptor Homo sapiens 4O9R Membrane 3.2 P1211 40.50, 157.30, 0.232, 2014a (Weierstall et al.,
52.40 0.278 2014)
90, 97, 90
Cry3A Bacillus thuringiensis 4QX0 Soluble 2.8 C2221 116.88, 134.81, 0.165, 2014a (Sawaya et al., 2014)
105.15 0.192
90, 90, 90
CPV17 polyhedrin Uranotaenia sapphirina 4S1K Soluble 2.2 I23 156.23, 89.29, 0.14, 0.19 2015a (Ginn et al., 2015)
cypovirus 96.42
90, 92.30, 90
Diacylglycerol kinase Escherichia coli 4UYO Membrane 2.18 P212121 75.30, 91.80, 0.208, 2014a (Li et al., 2015)
141.70 0.236
90, 90, 90
Xylose isomerase Streptomyces rubiginosus 4W4Q Soluble 2.0 I222 94.00, 100.00, 0.159, 2014b (Sugahara et al.,
103.00 0.196 2015)
90, 90, 90
Photoactive yellow protein Halorhodospira halophila 4WL9 Soluble 1.6 P63 66.90, 66.90, 40.80 0.198, 2014a (Tenboer et al.,
90, 90, 90 0.231 2014)
SR Ca2-ATPase Oryctolagus cuniculus 4XOU Membrane 2.8 C121 162.00, 76.30, 0.304, 2015a (Bublitz et al., 2015)
151.10 0.343
90, 109, 90
Angiotensin II receptor Homo sapiens 4YAY Membrane 2.9 C121 72.80, 41.00, 0.228, 2015a (Zhang et al., 2015)
167.70 0.274
90, 99.40, 90
Phycocyanin Thermosynechococcus 4Z8K Soluble 2.5 P63 153.40, 153.40, 0.187, 2015a (Conrad et al., 2015)
elongatus 39.64 0.255
90, 90, 120
Luciferin regenerating Photinus pyralis 5D9B Soluble 1.5 P212121 186.38, 186.38, 0.184, 2015b (Yamashita et al.,
enzyme 60.34 0.232 2015)
90, 90, 120
C-phycocyanin Thermosynechococcus 4ZIZ Soluble 1.75 H32 109.24, 109.24, 0.204, 2015a (Fromme et al.,
elongatus 452.64 0.254 2015)
90, 90, 90
Rhodopsin-arrestin Homo sapiens 4ZWJ Membrane, 3.3 P212121 48.21, 77.59, 84.76 0.252, 2015a (Kang et al., 2015)
complex soluble 90, 90, 90 0.293
Denotes experiments done at LCLS.
Denotes experiments done at SACLA.

around 10e30%, as higher crystals densities tend to lead to clog- owing jet [102]. In order to prevent dehydration, crystals must be
ging. Flowing jets remain the ideal method for sample delivery for immersed in oil before being painted onto the support grid.
most SFX experiments as they do not require freezing and are the Microuidic devices have also been used for xed target experi-
most practical technique for conducting time-resolved experi- ments at XFELs and have been designed to trap single crystals
ments. But for a small subset of experiments, xed targets may be [104,112]. These traps can theoretically ensure that the X-ray beam
benecial since this approach has the potential to decrease the interacts with only one crystal at a time but with current designs,
sample quantities necessary for a data set to be collected. Fixed crystal stacking will occur before all the traps are lled. Fixed tar-
targets vary from traditional goniometers, to windowed sample gets can result in crystal orientation bias [113]. Currently, xed
support wafers/grids (analogous to electron microscopy holders), target approaches are severely limited by slow data acquisition.
and to microuidic chips [45,104,106]. The main advantage of Data acquisition rates are primarily limited by the velocity of the
sample support wafers is their compatibility with 2D nanocrystals, xed target stage and the time it takes to replace the sample grid or
which require a scaffolding and thus, cannot be delivered in a chip. For example, if the stage motor could operate at 120 Hz and
J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47 41

automated scripts aligned the X-rays to each window/trap, a xed femtoseconds, which, within the framework of time-resolved
target containing 800 window/traps, the sample holder would crystallography, allow access to detect and resolve fast time
need to be replaced every 6.67 min. In addition, some xed targets points in catalytic reaction processes. Thereby, more temporally
produce high background due to the support material [103e105]. constrained intermediates can be detected along a reaction
Recently, data has also been collected at XFELs with a conven- pathway. Recent work has pushed temporal resolution to the sub-
tional goniometer approach on very large crystals (up to millime- picosecond regime [18,119], which is utterly out of reach at existing
ters in size) under cryogenic conditions. This method has been used synchrotron sources. This allows insight into ultrafast in-
to determine a dark structure of photosystem II with minimal X-ray termediates and therefore, the promise of a much ner under-
damage at 1.95 resolution [114]. However, this approach is low standing of catalytic mechanisms.
throughput and tedious as it can require days or even weeks of very With irreversible reactions one must consider the way in which
precious data collection time at FELs on hundreds of individually data is collected. In traditional crystallography a crystal is rotated
mounted crystals [111]. The experiment required freezing, attenu- during data collection and each diffraction pattern corresponds to a
ation, and translation of the beam focus by 50 mm at an X-ray spot rotational increment of the reciprocal space. If an irreversible re-
size of 1 mm to avoid X-ray damage. Furthermore, freezing and the action is to be probed fully, data collection would require thousands
large crystal size prohibits time-resolved experiments from being of large crystals for each time point as the crystal would be
feasible. Except for cases like photosystem II, where X-ray damage permanently altered after the induction of the reaction. This would
signicantly alters the structure of the catalytic metal complex, it is require an oppressively large number of crystals to collect a com-
questionable if this is advantageous compared to standard macro- plete data set from individual crystals. The serial diffraction before
molecular crystallography at synchrotron sources. destruction nature of SFX experiments completely bypasses this
constraint, as only one diffraction pattern is collected from each
4. Structural dynamics and molecular movies: challenges and crystal. Since the sample is constantly replenished, there is no
opportunities constraint on the reversibility of a reaction. The utilization of Monte
Carlo merging of an immense number of individual crystals in
In addition to broadening the array of macromolecules available random orientations leads to accurate structure factors for time-
to crystallography, SFX also expands upon the information that can resolved experiments due to implications from the central limit
be obtained regarding functional dynamics. Macromolecular crys- theorem.
tals typically exhibit extremely high solvent content and weak Considerations of structural homogeneity and the constraint of
electrostatic contacts in comparison to their smaller molecular Laue to predominantly pump-probe style experiments are highly
counterparts. While this has always presented a challenge to intertwined due to one parameter: diffusion. Thus far, Laue crys-
crystallogenesis of well-diffracting crystals, the porous nature of tallography has been predominantly limited to photo-activated
macromolecular crystals lend them to intact catalytic or functional reactions since chemically activated reactions would necessitate
activity in the crystalline phase [112e115]. This is because large diffusion of a substrate throughout the crystal, limiting reaction
solvent channels that are present in the crystals can allow for timescales available. While the large solvent channels in crystalline
conformational movement of the proteins or even the diffusion of a macromolecules often allow for diffusion to take place via soaking
substrate to active sites in crystals. This property indicates an with a diffusing substrate [120e122] (assuming the active site is
exciting opportunity to see intermediate structures over the course unobstructed and the substrate is sufciently small relative to the
of a functional reaction. By achieving time-resolved structure solvent channels), the size of the crystal limits diffusion times,
determination (i.e. multiple structures along a reaction pathway), thereby constraining the time regime of reaction intermediates
one can begin to directly understand the relationship between accessible in an experiment. There have been chemically activated
structure and function of macromolecules. This has numerous ap- time-resolved experiments performed successfully by incorpo-
plications such as rational drug design and renewable energy by rating photo-activated caged substrates into the crystal [123e125],
unraveling the mechanism of biochemical processes. However, thus reducing diffusion times to that of the photo-penetration of
some important considerations must be taken into account in order the pump laser. However, the incorporation of caged substrates
to realize reliable time-resolved structures. requires extensive knowledge about a given system and is not
Time-resolved crystallography has historically been performed generally compatible [126]. The small size of the crystals used in
on large single crystals by Laue multi-wavelength crystallography SFX theoretically allow for diffusion times on microsecond time
(for a review see [116]) and has exhibited considerable gains over scales, allowing access to many reactions on the short millisecond
the past twenty years, moving from the millisecond [117] to the and even microsecond regimes in the absence of caged substrates.
picosecond [118] temporal resolution regimes. This has opened the For example, a crystal with dimensions of 0.5  0.5  0.5 mm3 has
door to viewing conformationally dynamic proteins in action on an been modeled to exhibit a diffusion time of 17 ms, while a
atomic scale. While this technique continues to improve, both 3  4  5 mm3 crystal is estimated to take 1 ms, and a large
within its experimental parameters and subsequent data analysis, 300  400  500 mm3 crystal would take 9.5 s [126]. Many bio-
it faces some hard limitations due to the nature of large crystals and logical reactions occur with intermediates observed in the time
current synchrotron sources, namely: 1) temporal resolution is range of ms and faster but signicantly less occur in the longer
limited by pulse length achievable using a synchrotron, 2) irre- regime of seconds. One must also consider reaction homogeneity
versible reactions cannot be studied as the induction of a reaction since Bragg diffraction relies upon translational symmetry of the
would cause a permanent modication of the molecules in the molecules in the crystal. This means that in order to observe an
crystal, 3) only light activated, pump-probe type experiments are intermediate structure, a sufcient proportion of the molecules
generally feasible at present, with limited light penetration being must be in a single conformation during probing in order for a
one of the major obstacles, and 4) homogeneity of reaction initia- structure to be elucidated.
tion must be considered and presents a challenge. Photoactivated reactions have one major advantage over sub-
SFX presents an opportunity to complement the Laue method strate or other diffusion based reactions as initiation is homoge-
by providing access to experiments previously impossible by neous and rapid. However, the degree of reaction initiation by
overcoming the above challenges via its unique experimental photoinduction is also limited by the size of the crystal since
characteristics. XFELs have pulse durations on the order of tens of molecules absorb the light as it travels through the crystal causing a
42 J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47

decrease in transmission with increasing path length. When states to be studied along the multiple-excitation reaction pathway.
considering faster time points, the reaction homogeneity of the This revealed large conformational changes occurring in the photo-
molecules in the crystal is sensitive to the lifetime of the inter- excited double ash state of PSII, which includes movement of
mediate state as compared to the time difference of reaction initi- the protein via its coordination of the oxygen evolving cluster [17].
ation from the front and back surfaces of the crystal relative to the Though higher resolution is needed to provide a deeper under-
pump beam. The distance traveled with respect to the front and standing of the water splitting process, TR-SFX in general provides
back of photo-activated crystals is not the main cause of this tem- the most feasible path forward toward obtaining a series of initial,
poral offset (though this is on the order of picoseconds for large nal and intermediate structures during a reaction, i.e. molecular
crystals); instead, it is the attenuation of the pump intensity as it movies of biomolecules at work.
proceeds through the crystal, undergoing absorption and scat- More recently, atomic resolution has been achieved for TR-SFX
tering. Depending on the particular robustness of the sample, one using PYP [16]. This experiment also pushed the limits on the
can attempt to address this by increasing the power of the laser but time-resolution available at an XFEL, capturing reaction time points
this approach is constrained by photo-damage and heating effects down to the sub-picosecond regime [119]. These studies improve
that can occur. Thus, the size of nanocrystals provides an advantage, upon the temporal resolution that has been achieved in Laue
allowing a much smaller difference of reaction initiation crystallography [118] by orders of magnitude and is only signi-
throughout the crystal and requiring decreased pump intensities cantly limited by beam characteristics (i.e. time-jitter) [119]. As
for maximal reaction initiation. Furthermore, the volume of the developments continue with future generation sources, temporal
crystal itself is so small that in practice, the whole crystal can be resolution will be pushed even further. In addition to ultrafast time
illuminated by the pump, minimizing considerations of the in- resolution, it is notable that the TR-SFX studies on PYP have also
tensity prole of the pump laser. Because of these attributes, SFX shown huge improvement on reaction initiation (40% at XFELs with
provides the basis for a much higher reaction initiation yield, a nanocrystals compared to 10e15% achieved at a synchrotron with
point which as has already been shown in practice [16]. larger crystals [15,16]), allowing for stronger signal from transient
intermediates to be detected, leading to data that can be easily
4.1. Time-resolved serial femtosecond crystallography interpretable. Even more recently, research in this time domain has
led to novel observations of global structural changes in myoglobin
The structure-mechanistic relationship found in biological re- upon lysis of an Fe-CO bond that occur within a few picoseconds
actions is rarely explained through resolution of a single, static [18].
structure. Instead, the dynamics between initial and nal states Though no mixing TR-SFX work has been published to date, the
embody the mechanism. TR-SFX permits access to transient in- stage is set for on the y mixing experiments that take advantage
termediates that occur as a reaction is proceeding, providing still of the nature of SFX. Many enzymes have biologically interesting
snapshots of these states. Snapshots of the initial, intermediate, and intermediates that occur on time scales faster than those accessible
nal state(s) can be viewed in a quick succession from start to nish to soaking experiments of large crystals. Although one could scale
to reveal motion of the enzyme and thus a molecular movie can down the size of the crystals and thereby, narrow the reaction
be obtained to unravel how these biological molecules proceed in initiation temporal prole, the tradeoff with decreased diffraction
nature. severely limits the range of this method. In theory, the sizes of
The rst time-resolved SFX (TR-SFX) experiment was carried out crystals available for SFX alleviates this concern, decreasing
on PSI-ferredoxin co-crystals which undergo electron transfer re- necessary mixing times such that a new regime of reactions is
actions that lead to the subsequent undocking of ferredoxin and accessible. Delivery methods based on the GDVN have already been
dissolution of the crystals upon light excitation, leading to a rapid developed for time-resolved mixing experiments e.g. a double
loss of diffracting quality upon pumping. Large differences have focused mixing jet [19] where a jet containing crystals is mixed
been obtained in the diffraction patterns between the excited and within a stream containing the desired substrate prior to dis-
uninitiated reactions as revealed by the Wilson plot. The timing of charging from the nozzle. For a more in depth look at substrate
these differences agrees with the time range previously found by mixing techniques, the reader is referred to [126].
spectroscopic methods for electron transfer [127,128]. These ex- Indeed, the most promising areas for XFELs to push the
periments provided the proof of concept for TR-SFX, (though an boundaries within structural biology is the ability to study tem-
electron density map was not obtained due to data limitations) poral dynamics with femtosecond pulses, allowing access to ul-
[41]. trafast timescales and thereby, short lived intermediates. Without
The rst successful TR-SFX experiment was performed using sacricing resolution, TR-SFX paves the way for dynamic structural
photosystem II, the membrane protein responsible for splitting elucidation of biological processes that feature fast (<100 ps)
water into its constituent protons, electrons and oxygen. This conformational changes, irreversible reactions, non-photo-
process provides the electrons for the photosynthetic electron activated inductions, and to those which are limited by crystal
transfer chain in oxygenic photosynthesis. The catalytic oxygen size. TR-SFX can also avoid the local radiation damage often expe-
evolving cluster, structurally the most interesting domain in rienced in the active sites of macromolecules and generate a higher
photosystem II, is particularly susceptible to radiation damage due fraction of intermediate states [16]. This has far reaching implica-
to the presence of 5 Mn atoms. The X-ray photo-damage processes tions for many elds, notably alternative energy and drug design,
are virtually eliminated by the diffraction before destruction na- which both rely heavily on understanding the relationship between
ture of SFX. A further concern of time-resolved studies with X-rays structure and mechanism. The reactions accessible to TR-SFX
is that local photo-damage can be induced by repeated illumination studies will continue to broaden as mix and inject methods for
of the crystal by the pump laser in Laue time-resolved experiments. chemo-activated reactions continue development. With TR-SFX
Again, this problem is mitigated by TR-SFX since relatively low and other time-resolved XFEL methods (such as time-resolved
pump energy and ux can be applied per crystal due to smaller, wide angle X-ray scattering, e.g. [129]), it is evident that XFELs
more permeable crystals. Indeed, TR-SFX has succeeded in shining provide new ways to explore a novel regime of time-resolved
the rst light on the undamaged ground state of PSII using single structural biology, leading towards true movies of dynamic mac-
and multiple laser excitations prior to diffraction, allowing multiple romolecules in action.
J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47 43

5. Data acquisition and data processing in SFX then subjected to Bragg peak location analysis (so called peak
nding) in which Bragg peaks are identied by searching for
The short duration pulses delivered by XFELs have necessitated clusters of connected pixels based on a series of parameters
the development of new detector technologies capable of inte- including minimum numbers of pixels per peak, the number of
grating all of the photons that arrive within the time duration of a peaks in a frame, intensity thresholds, and signal to noise ratio. A
few femtoseconds, while sustaining full-frame readout at the XFEL minimum number of Bragg peaks must be identied in the
pulse repetition rate. Although X-ray charge coupled device (CCD) diffraction pattern as a further constraint, so that patterns with too
detectors are very common at synchrotrons, few CCD detectors few peaks (i.e. impossible to index) are rejected.
have readout speeds that match the LCLS repetition rate. Two CCD's The background corrected and sorted diffraction patterns
were used at LCLS for the rst experiments (at 2 keV), the pnCCD identied during the hit nding process are subjected to indexing
and the fCCD [130,131]. Due to their very low noise and high and integration by programs such as CrysFEL [136], cctbx.xfel [101],
quantum efciency over a large range of energies, they have been and nXDS [139]. The purpose of these programs is to identify Bragg
used for imaging and spectroscopy experiments. However, a larger peaks in the hits and then to perform indexing. Determining the
dynamic range then the CCDs can currently cover (a few hundred unit cell parameters and the orientation of a crystal is carried out by
thousands of photons) is necessary for SFX experiments at atomic the widely used algorithms such as MOSFLM [140e142], DIRAX
resolution (energies >6 keV). The rst detector specically [143], LABELIT [144], and XDS [139], based on the Bragg peak lo-
designed for higher energies presently used for SFX experiments at cations in a diffraction pattern. Once each pattern has been suc-
LCLS was the Cornell-SLAC pixel array detector (CSPAD) [132e135]. cessfully indexed, the intensities are merged and integrated using
Today the CSPAD is the principal detector used for SFX experiments the Monte Carlo method [53,145]. One of the largest problems in
at LCLS. It consists of 64 separate modules (194  185 pixels each), SFX data analysis is the indexing ambiguity which occurs when the
allowing cost-effective replacement and experimental exibility. Bravais symmetry is higher than the space group symmetry. An
The CSPAD is tiled to produce a 2.3 megapixel detector, with indexing ambiguity arises in some polar space groups (like P63)
readout speeds matching the repetition rate of 120 Hz [133]. The where each pattern has multiple ways it can be indexed. In stan-
panel distribution leaves an adjustable sized hole in the middle to dard crystallography this indexing ambiguity is solved by explo-
allow for adjustable incident beam focuses, preventing the beam ration of the data set with both indexing options, where only one
from damaging the detector (the beam is powerful enough to melt option leads to a correct X-ray structure. However, since many in-
through a conventional beam stop) [136]. dividual patterns are merged in SFX, the ambiguity must be solved
The use of detectors composed of multiple modules introduces before any additional processing to avoid articial twinning. In
unique concerns, namely the exact location of each module must be order to overcome the indexing ambiguity problem, CrystFEL has
known in order to correctly assess the data. While the experimental implemented an algorithm based on the expectation maximization
geometry may be known to low precision prior to an experiment, it approach [146] which has been successfully applied and validated
can be subsequently rened using the collected data. In this case, using both simulated and experimental diffraction [146].
assembling a physically correct image during initial processing is As with conventional crystallography, the phase problem has
futile and a known calibration sample is used to rene the exper- to be solved in order to reconstruct a real-space electron density
imental geometry. The detector geometry is specied in a pixel map from the measured SFX intensities. Indeed, this presents itself
location map containing the coordinates of each detector pixel in a as a primary challenge in serial crystallography data analysis. Until
suitably dened coordinate system. All constraints for an experi- recently, all crystallographic structures so far determined by XFEL
mental geometry description are saved in a single text le which have been phased by molecular replacement, using phases from
can be implemented within any of the available SFX software known or related structures [11,32,33,47,51]. This method limits the
[101,136,137]. The success of indexing, predicting spot locations target protein molecules to be investigated by SFX since the protein
using a crystal orientation matrix and integrating reection in- of interest must have a known homologous structure. Even if it
tensities depends upon the precise knowledge of the location of does, there is risk that phase bias can be introduced. However, some
these sensors in three-dimensional space. This means that an ac- conventional phasing methods have also been suggested to be
curate calibration and renement of the tile metrology is critical. applied to SFX data such as multiple isomorphous replacement or
Data analysis in SFX has unique challenges with respect to data multi/single-wavelength anomalous dispersion (MIR, MAD, SAD)
sets collected at synchrotrons. This is due to the serial nature from [18,42,147,148]. Barends [42] and co-workers have demonstrated
snapshot diffraction patterns of randomly oriented crystals with for the rst time that the conventional phasing method of SAD can
unknown partiality and shot to shot variation in the beam char- also be successfully used for SFX experiments [42]. In this experi-
acteristics. Furthermore, the images collected during an experi- ment they collected and solved the structure to 2.1 resolution of a
ment consist not only of single crystal hits (one crystal in the beam) lysozyme heavy atom derivative that gives a strong anomalous
but also blank patterns (no crystal in the beam) and multi-hits signal from two gadolinium atoms per asymmetric unit. The MAD
(multiple crystals in the beam). The XFELs at LCLS and SACLA phasing method has also been recently adapted to SFX by using
operate up to 120 Hz and 60 Hz, respectively, resulting in hundreds modied Karle-Hendrickson equations [147,148]. This proposed
of thousands of patterns collected per hour, thus creating terabytes generalized version of MAD phasing method offers another po-
of data. Due to these challenges, conventional crystallographic data tential for experimental phasing for structural determination in
processing methods cannot be efciently used for SFX data SFX.
collection. Thus, new data analysis tools for SFX have been devel- In addition to the classical methods, new phasing techniques
oped. In order to optimize efciency during data collection it is have been proposed for SFX data analysis. Methods such as ab initio
important to have rapid feedback during data collection. First, data phasing by the evaluation of the shape transforms (or the over-
is reduced by eliminating blank and multi-hit patterns which is sampling method) [11,40,149,150] and the high-intensity radiation
implemented within the programs Cheetah [137] and CASS [138]. induced phasing (HI-RIP) [92] have been proposed. The phase
They perform the data pre-processing steps, evaluate the quality of transform method exploits the intensity scattered between
each data frame, and reject all those frames that are not suitable for neighboring Bragg peaks (or fringes) from crystals that contain less
further analysis. Once the data size is reduced, detector artifacts are than 20 unit cells in each crystal direction. This phenomenon has
removed and background subtraction is performed. Each frame is historically been obscured by noise in large crystals due to inverse
44 J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47

scaling of inter-Bragg intensity with the number of unit cells. Each coherence) have attracted considerable attention of a wide com-
recorded Bragg peak represents a particular portion of the Ewald munity of scientists in elds ranging from material science, to
sphere and phasing is achieved by oversampling between the Bragg chemistry, to structural biology, and to high energy physics. This
peaks. The HI-RIP method takes advantage of the ionization process has led to ground breaking discoveries but new science brings new
of the atoms that occur within the femtosecond timescale of the challenges and access to experimental time at XFELs is presently
pulse in SFX experiments [151]. The change in the electronic one of the major limitations. Currently, there are only two high
conguration of atoms leads to the modication of the atomic energy XFELs in operation in the world (i.e. only two experiments
scattering factors during diffraction [148]. In the case of SFX this can take place in the world at the same time). It is exceedingly
would introduce the possibility of determining phases by varying unfortunate that due to this, beamtime is so scarce, thus becoming
the scattering factors of the heavy atoms. This new approach, which a major limiting factor as the eld progresses out of its initial stages.
has been already tested with Trypanosoma brucei cathepsin B pro- Fortunately, three new XFELs are currently under construction, in
tein, would represent a powerful method of experimental phasing South Korea (PAL), in Switzerland (SwissFEL), and in Germany
without the need to modify the protein crystals assuming sulfur (European XFEL), which are expected to enter the commissioning
atoms are present in the native structure [92]. phase in 2016 (PAL) and 2017 (European XFEL and SwissFEL).
Furthermore, XFEL facilities are planned or are under initial stages
6. Challenges and outlook of construction in Italy and China.
Along with the unprecedented new scientic opportunities, the
Nearly six years after the rst SFX data were collected using an successes of LCLS and SACLA have opened the eyes of the com-
XFEL at LCLS and four years after the proof of principle of SFX was munity for the need for further novel instrumentation de-
published [11], a new era in structural biology has emerged. Fig. 5 velopments in the eld of XFELs. One challenge is the production of
shows a gallery illustrating the breadth of protein structures suc- coherent photons of higher energies (50 keV or more). Another is
cessfully solved with XFELs to date. The unique properties of XFELs the production of single spike pulses that are shorter than 1 fs. In
(ultrashort, extremely intense pulses with high frequency and addition, optics, diagnostics, detectors, sample delivery, and data

Fig. 5. Select structures solved using XFELs by PDB code. From top left: photosystem I (3PCQ [11]), FABP3 (3WXQ [61]), purple bacterial reaction center (4CAS [46]), lysozyme (4ET8
[32]), cathepsin B (4HWY [51]), 5-HT2B (4NC3 [27]), photosystem II (4PBU [17]), d-opiod receptor (4RWD [44]), smoothened receptor [35], Cry3A (4QX0 [52]), CPV17 polyhedron
(4S1K [68]), diacylglycerol kinase (4UYO [102]), xylose isomerase (4W4Q [61]), photoactive yellow protein (4WL9 [16]), SR Ca2-ATPase (4XOU [58]), angiotensin II receptor (4YAY
[76]), phycocyanin (4Z8K [65]), luciferin-regenerating enzyme (5D9B [60]), C-phycocyanin (4ZIZ [66]), rhodopsin-arrestin complex (4ZWJ [48]).
J.M. Martin-Garcia et al. / Archives of Biochemistry and Biophysics 602 (2016) 32e47 45

acquisition must continue to be developed to keep up with the new To support next generation XFEL facilities, new features are
development in data acquisition speed and X-ray pulse duration continually under development to meet the constantly evolving
and intensity. Lower noise and higher dynamic range detectors are needs of new experiments. In fact, while originally designed for
needed to take full advantage of these scientic opportunities. In implementation at LCLS, Cheetah has undergone several updates in
this sense, a second generation of XFEL-capable detectors, the ePix the last year and a half and has now been implemented in serial
family, is being developed at SLAC for this purpose. In parallel with millisecond crystallography experiments at synchrotron sources
the development of second generation XFELs, a new generation of ( [97]) and for SFX data
detectors are being developed to meet the technical specications collected at SACLA (
required (such as the AGIPD [152] at European XFEL or the Jungfrou commits/online). In addition to Cheetah and CASS software, a
at SwissFEL) [153]. new software system called Karabo is currently under development
Research in the eld of structural biology can now collect nearly at the European XFEL. European users will have access to CrystFEL
damage-free X-ray data on biomacromolecular nanocrystals and through Karabo to enable fast data analysis required by the
continues to greatly impact the eld by addressing many limita- immense data acquisition challenge.
tions faced by traditional crystallography. Going beyond SFX, With proof of principle experiments already displaying the
computational simulations using rubisco have shown that a serial breadth of the technique, it is clear that the future is not just bright
femtosecond imaging technique on individual molecules, in com- but brilliant. As SFX emerges from its infancy, it is apparent that
bination with the oversampling phasing method, could open a new molecular movies will provide a bedrock for new advances and
horizon of structural elucidation of macromolecules without the discoveries from structural biology, to medicine, to energy
need to rst crystallize them [154]. However, with the photon ux conversion.
of current XFELs, the ultimate goal of collecting atomic resolution
data on a solution of individual, non-crystallized molecules re- Acknowledgements
mains currently out of reach. As the technique progresses, single
particle imaging to atomic resolution may become feasible with This work was supported by the STC Program of the National
future development of XFELs and data processing. In order to obtain Science Foundation through BioXFEL under Agreement No.
high resolution 3D structural information of single large protein 1231306, the National Institutes of Health Femtosecond Nano-
complexes, there are several challenges that need to be overcome. crystallography of Membrane Proteins Award 617095583, the
First, the pulse uence of XFELs is not yet high enough to allow the PSI:Biology Center MPID U54GM094625, and the Center for
measurement of high resolution diffraction signal from a single Applied Structure Discovery.
large protein complex. This can, in principle, be overcome by
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