research papers

IUCrJ

Serial femtosecond crystallography of soluble
proteins in lipidic cubic phase

ISSN 2052-2525

BIOLOGY j MEDICINE

Received 1 May 2015
Accepted 8 July 2015

Edited by Z.-J. Liu, Chinese Academy of
Sciences, China
Keywords: serial femtosecond crystallography;
X-ray free-electron laser; lipidic cubic phase;
soluble protein.
PDB references: lysozyme, 4zix; phycocyanin,
4ziz
Supporting information: this article has
supporting information at www.iucrj.org

Raimund Fromme,a Andrii Ishchenko,b Markus Metz,c,d Shatabdi Roy Chowdhury,a
Shibom Basu,a Sébastien Boutet,e Petra Fromme,a Thomas A. White,c Anton Barty,c
John C. H. Spence,f Uwe Weierstall,f Wei Liua* and Vadim Cherezovb*
a
Center for Applied Structural Discovery at the Biodesign Institute, Department of Chemistry and Biochemistry,
Arizona State University, 727 East Tyler Street, Tempe, AZ 85287, USA, bBridge Institute, Department of Chemistry,
University of Southern California, 3430 South Vermont Avenue, MC 3303, Los Angeles, CA 90089, USA, cCenter for Free
Electron Laser Science, Deutsches Elektronen-Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany,
d
Department of Physics, University of Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany, eLinac Coherent
Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA, and
f
Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287, USA. *Correspondence e-mail:
w.liu@asu.edu, cherezov@usc.edu

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs)
enables high-resolution protein structure determination using micrometre-sized
crystals at room temperature with minimal effects from radiation damage. SFX
requires a steady supply of microcrystals intersecting the XFEL beam at random
orientations. An LCP–SFX method has recently been introduced in which
microcrystals of membrane proteins are grown and delivered for SFX data
collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic
phase (LCP), using a special LCP microextrusion injector. Here, it is
demonstrated that LCP can also be used as a suitable carrier medium for
microcrystals of soluble proteins, enabling a dramatic reduction in the amount
of crystallized protein required for data collection compared with crystals
delivered by liquid injectors. High-quality LCP–SFX data sets were collected for
two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each
protein.

1. Introduction
The recent advent of hard X-ray free-electron lasers (XFELs)
has opened up many exciting opportunities in structural
biology (Feld & Frank, 2014). Intense XFEL pulses of
extremely short duration (<50 fs) make it possible to outrun
radiation damage, as predicted by Neutze et al. (2000) and as
later demonstrated by Chapman et al. (2006), enabling structure determination from tiny crystals at room temperature.
Data are usually acquired using a serial femtosecond crystallography (SFX) approach, in which a continuous stream of
microcrystals is intersected with the XFEL beam and
diffraction patterns from individual crystals at random orientations are recorded at the pulse repetition rate of the laser
(Chapman et al., 2011). Following the automated indexing and
merging of thousands of diffraction patterns, a Monte Carlo
method is used to integrate over the angular profile of the
Bragg reflections and other stochastic fluctuations (Kirian et
al., 2011). SFX has been successfully applied to both soluble
proteins (Boutet et al., 2012; Redecke et al., 2013; Sawaya et al.,
2014) and membrane proteins (Johansson et al., 2012; Kern et
al., 2013; Johansson et al., 2013) using crystals ranging in size
from nanometres to micrometres. These were formed in either
aqueous solution or lipidic sponge phase and continuously
delivered to the XFEL beam through a fast-running liquid
microjet produced by a gas dynamic virtual nozzle (GDVN)
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injector (DePonte et al., 2008). Initial proof-of-concept
experiments have shown the potential of this technology for
structural studies of proteins that are refractory to the growth
of sufficiently large, high-quality crystals suitable for data
collection at synchrotron sources, as well as for time-resolved
studies of unstable intermediate states and irreversible
processes. This technique, however, requires very large
amounts (10–100 mg) of crystalline material for data collection, most of which runs to waste between shots, limiting its
use to only well expressed and well behaved proteins. To
address this issue, we have previously introduced an LCP–
SFX method (Liu et al., 2013, 2014; Weierstall et al., 2014) in
which membrane proteins are crystallized and delivered for
data collection inside a gel-like lipidic cubic phase (LCP). The
specific texture and high viscosity of the LCP medium allows a
reduced flow rate compared with the GDVN injector, so that
the crystals are used more efficiently. Using LCP as a carrier
medium thus greatly reduces the amount of protein required
to collect a complete set of diffraction data.
Apart from membrane proteins, which can be crystallized in
LCP and are amenable to LCP–SFX, there are many important soluble macromolecules that represent challenging crystallization targets (Garman, 2014). These include large protein
complexes, protein–DNA and protein–RNA complexes and
proteins with dynamic domains. Quite often, limited quantities
of sub-10 mm crystals of such macromolecules are available;
however, obtaining large crystals suitable for data collection at
synchrotrons or producing large quantities of microcrystals for
SFX using a liquid injector may represent formidable obstacles. Here, we have modified our LCP–SFX protocol to
demonstrate that LCP can serve as a suitable medium for the
efficient delivery of soluble protein crystals for crystallographic data collection at XFELs.

2. Methods
2.1. Lysozyme microcrystal sample

Chicken egg-white lysozyme powder (Sigma; catalog No.
62970) was dissolved in 0.02 M sodium acetate pH 4.6 buffer
to a final concentration of 50 mg ml1. Crystallization was
initiated by injecting 20 ml lysozyme solution (50 mg ml1)
into 1 ml precipitant solution [20%(w/v) NaCl, 6%(v/v) PEG
6000, 1 M sodium acetate pH 3.0] at room temperature.
Lysozyme microcrystals with average dimensions of 5  2 
2 mm formed immediately upon vortexing the resulting solution.
The suspension of lysozyme microcrystals was centrifuged
at 500g for 5 min. The supernatant (precipitant; around 980 ml)
was carefully removed and the remaining 40 ml of lysozyme
microcrystal/precipitant solution was used for LCP preparations.
Finally, the concentrated suspension of lysozyme crystals
(lysozyme concentration of 25 mg ml1) was mixed with
lipids (1:1 9.9 MAG:7.9 MAG by weight) in a volume ratio of
45:55 using a dual-syringe lipid mixer until a homogeneous
LCP formed (Cheng et al., 1998; Caffrey & Cherezov, 2009).

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Table 1
XFEL data-collection and refinement statistics for lysozyme.
Values in parentheses are for the highest resolution shell.

Data collection
Average crystal dimensions (mm)
Amount of protein used (mg)
Wavelength (Å)
Pulse duration (ns)
Beam size (mm)
Flux (photons per pulse)
Maximum dose per crystal (MGy)
Space group
Unit-cell parameters (Å)
No. of collected images
No. of hits/indexed images
Total/unique reflections
Resolution (Å)
Completeness (%)
Multiplicity
hI/(I)i
CC1/2
Rsplit (%)
Refinement
No. of reflections
No. in test set
Rwork/Rfree (%)
No. of atoms
Protein
Water and others
B factors (Å2)
Wilson/overall
Protein
Water and others
R.m.s.d., bonds (Å)
R.m.s.d., angles ( )
Ramachandran plot statistics (%)
Favored
Allowed
Disallowed

Lysozyme,
LCP injector

Lysozyme,
GDVN injector†,
reprocessed

225
0.1
1.56
35
1.5
5  109
2.5
P43212
a = b = 79.1,
c = 38.6
299569
119844/54544
27923651/10266
27.97–1.89
(1.96–1.89)
100.0 (100.0)
26558 (271.4)
9.5 (1.9)
99.1 (68.1)
8.2 (51.4)

112
15
1.32
40
3
4  1011
33
P43212
a = b = 78.9,
c = 37.9
1512940
69621/24465
10413069/10006
35.26–1.90
(1.97–1.90)
100.0 (100.0)
10407 (933.9)
8.9 (4.3)
98.9 (93.7)
8.9 (20.8)

10266 (948)
520 (53)
16.5 (25.2)/
19.1 (25.1)

9856 (892)
500 (52)
16.5 (16.1)/
19.8 (24.7)

1015
93

1001
90

28.6/29.8
29.0
37.0
0.003
0.71

24.3/25.3
24.50
34.50
0.010
1.2

99.2
0.8
0

99.0
1.0
0

† Data from Boutet et al. (2012).

2.2. Phycocyanin microcrystal sample

Thermosynechococcus elongatus cells were preprocessed
with a microfluidizer to break the cell walls. This was followed
by a series of centrifugation cycles to isolate the thylakoid
membrane. Phycobiliproteins such as phycocyanin (PC) and
allophycocyanin (APC) were isolated by ultracentrifugation
of the supernatant obtained after microfluidizer treatment.
Cell debris and larger particles were spun down at 50 000g for
1 h. The supernatant was concentrated using Centricon spin
filters with a molecular-weight cutoff of 100 kDa to obtain
concentrated protein. PC microcrystals were produced by
the free-interface diffusion method (Kupitz et al., 2014) at a
starting concentration of 50 mg ml1 and using 75 mM
HEPES pH 7, 20 mM MgCl2, 17% PEG 3350 as the precipitant. The final concentrations of protein and PEG 3350 were
half of the starting values. The procedure of LCP sample
preparation was identical to that described above. PC microcrystals with average dimensions of 10  10  5 mm were
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Table 2

geometry were optimized to
ensure the best quality of peak
Values in parentheses are for the highest resolution shell.
finding and indexing. Autoindexing and structure-factor
PC, macrocrystal
integration of the crystal hits
(PDB entry 3l0f)
PC, GDVN injector
PC, LCP injector
was performed using CrystFEL
Data collection
(White et al., 2012), which
Average crystal dimensions (mm)
200  200  300
10  10  5
10  10  5
Amount of protein used (mg)
N/A
30
0.1
involved the application of fast
Wavelength (Å)
1.00
1.45
1.56
Fourier transform (FFT)-based
Maximum dose per crystal (MGy) N/A
N/A
66
autoindexing algorithms, MOSFLM
Space group
H32
H32
H32
Resolution (Å)
19.0–1.35 (1.42–1.35)
36.4–1.95 (2.02–1.95)
31.6–1.75 (1.81–1.75)
(Leslie, 2006), DirAx (DuisenUnit-cell parameters (Å)
a = b = 187.1, c = 59.8 a = b = 186.4, c = 60.3 a = b = 187.1, c = 60.5
berg, 1992) and XDS (Kabsch,
No. of hits/indexed images
N/A
36118/16689
18794/6629
2010) followed by averaging and
Total/unique reflections
600252/86960
7520260/32291
6171418/44284
hI/(I)i
19.15 (2.35)
2.92 (1.44)
2.94 (1.33)
integration of Bragg peaks using a
Multiplicity
7.0 (5.9)
358.6 (47.9)
139.4 (36.8)
Monte Carlo algorithm. The final
Completeness (%)
99.9 (99.7)
99.98 (99.8)
99.97 (100)
statistics of the quality of the data
CC1/2
0.999 (0.799)
0.975 (0.34)
0.951 (0.32)
Rsplit (%)
NA
31.5 (97.3)
39.1 (94.2)
sets are summarized in Tables 1
Rmerge (%)
7.7 (84.9)
N/A
N/A
and 2. The maximal radiation
Refinement
dose per crystal was estimated
No. of reflections
82594 (6097)
32286 (3188)
40257 (4000)
No. in test set
4367 (296)
1660 (176)
1838 (183)
using RADDOSE (Paithankar et
Rwork/Rfree (%)
13.6 (27.1)/17.5 (33.1) 24.0 (37.5)/28.7 (41.9) 20.4 (46.9)/25.2 (50.2)
al., 2009). (I) values were estiNo. of atoms
mated as the standard deviations
Protein
2497
2497
2497
Water and others
573
258
300
of the means of the intensity
B factors (Å2)
measurements (White et al.,
Wilson/overall
16.3/16.5
15.3/24.1
33.8/37.5
2012). The appropriate resolution
Protein
20.7
23.9
37.0
Water and others
41.5
27.8
45.6
cutoff was based on the behavior
R.m.s.d., bonds (Å)
0.007
0.024
0.012
of the Pearson correlation coeffi
R.m.s.d., angles ( )
1.58
1.29
1.24
cient CC1/2 versus resolution, and
Ramachandran plot statistics (%)
Favored
98.2
98.2
98.5
on the improvements in the Rwork/
Allowed
1.8
1.8
1.5
Rfree values after including higher
Disallowed
0
0
0
resolution shells in refinement
(Karplus & Diederichs, 2012).
After integration with CrystFEL,
grown at 4 C over 9–14 h and pooled together before mixing
the initial phases were obtained by molecular replacement
with LCP as described above for the lysozyme samples.
using known structures of the protein from the PDB (PDB
entries 4et8 for lysozyme and 3l0f for PC; Boutet et al., 2012;
R. Fromme, D. Brune & P. Fromme, unpublished work) and
2.3. XFEL data collection and treatment
the structures were refined using phenix.refine (Afonine et al.,
2012), including several simulated-annealing cycles in order to
Experiments were performed using the CXI instrument
reduce phase bias. Structure images were prepared using
(Boutet & Williams, 2010) at the Linac Coherent Light Source
PyMOL (Schrödinger). The final coordinates and structure
(LCLS) at SLAC National Accelerator Laboratory. LCLS
factors were deposited in the PDB under accession codes 4zix
was operated at a wavelength of 1.56 Å (7.95 keV), delivering
(lysozyme) and 4ziz (PC).
individual X-ray pulses of nominally 35 fs pulse duration.
Protein microcrystals in LCP medium were injected at an
average flow rate of 170 nl min1 into the XFEL beam focus
3. Results
region inside a vacuum chamber using an LCP injector with a
50 mm diameter nozzle. Single-shot diffraction patterns of
We have collected full XFEL diffraction data sets from two
randomly oriented crystals were recorded at 120 Hz with a
soluble proteins as model systems: the small, 14.3 kDa,
Cornell–SLAC Pixel Array Detector (CSPAD) positioned at a
chicken egg-white lysozyme and the relatively large, heterodistance of 100 mm from the sample (Hart et al., 2012).
hexameric, 120 kDa, phycocyanin disk-like complex involved
In the case of lysozyme, a total of 299 569 images were
in light harvesting in photosynthesis as part of a phycobilisome
collected within 45 min, of which 119 844 were identified as
in cyanobacteria. The proteins were first crystallized in their
crystal diffraction patterns by Cheetah (Barty et al., 2014),
corresponding crystallization buffers, after which the slurries
corresponding to an average hit rate of 40%. In the case of PC,
of microcrystals were concentrated by centrifugation to the
a total of 287 520 images were collected within 40 min, of
desired concentration and mixed with appropriate LCP host
which 18 794 were identified as crystal hits (average hit rate of
lipids (Figs. 1a and 1b) using a lipid syringe mixer as described
6.5%). The peak-detection parameters and the experimental
previously (Liu et al., 2014). Mechanical mixing allows the fast
XFEL data-collection and refinement statistics for PC.

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and efficient formation of LCP; however, the shear forces can
damage or dissolve the crystals if mixing is too vigorous. We
observed that with gentle mixing, crystals over 10–20 mm in
size, especially those with needle or plate-like shapes, tended
to break into smaller pieces of less than 10 mm in size which, in

turn, are sturdy enough to withstand the mechanical stress
associated with mixing. Such crystal breakage did not affect
the diffraction properties of the crystals in our test samples.
The resulting dispersion of microcrystals in LCP was then
transferred to an LCP injector and diffraction data (Figs. 1c
and 1d) were collected at 120 Hz at the
LCLS using an LCP flow rate of
170 nl min1 as described previously
(Liu et al., 2013; Weierstall et al., 2014).

3.1. Lysozyme structure

Figure 1
Representative crystal images and diffraction patterns. (a, b) Pictures of crystals embedded in LCP
for lysozyme (a) and PC (b). (c, d) Full diffraction patterns for lysozyme (c) and PC (d). Parts of the
diffraction images, outlined by squares in (c) and (d), are enlarged in (e) and ( f ), respectively, to
facilitate visualization of individual diffraction peaks. Diffraction peaks identified by Cheetah are
circled.

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The structure of lysozyme was solved
at 1.9 Å resolution using 54 544 indexed
single-crystal diffraction snapshots. The
resulting overall structure is very similar
to the previously published lysozyme
structure determined by SFX using a
GDVN injector (Boutet et al., 2012).
Since the software has substantially
advanced over the last two years, we
have also reprocessed the data from
Boutet et al. (2012) using more recent
versions of Cheetah (v.2013.3) and
CrystFEL (v.0.5.3a), resulting in
considerably improved data statistics.
Both data sets were processed in the
same way using the same versions of
the software to avoid bias (Table 1). We
compared the structures of lysozyme
obtained using both LCP and GDVN
injectors to ensure the validity of our
procedure. The structures aligned very
closely, with an r.m.s.d. of only 0.5 Å.
The B-factor distributions are very
similar, with a slightly higher average B
factor in the case of the LCP structure.
The quality of the maps is also very
similar, with no structural differences
observable in difference maps (Figs. 2a
and 2b). Small differences were only
detected around the side chains of
solvent-exposed bulky amino-acid residues. These minor differences can be
expected given the variations in the
crystal-preparation protocols, crystal
size and crystal-delivery methods. We
can therefore conclude that using LCP
as a carrier medium for SFX data
collection results in a similar quality
structure compared with the previously
used GDVN injector, while offering the
important advantage of consuming
considerably less crystallized protein
(0.1 mg using the LCP injector versus
15 mg using the GDVN injector).
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Figure 2
Lysozyme structure. (a) Overall structure of lysozyme showing 2mFo  DFc electron density around several residues in the active site contoured at 1.
(b) Difference electron density between the data collected in this work and those presented in Boutet et al. (2012), contoured at 3. Positive difference
density is green and negative is red.

3.2. Phycocyanin structure

As the second test protein, microcrystals of phycocyanin
(PC), a photosynthetic pigment protein from the thermophilic
cyanobacterium T. elongatus, were used. SFX data were
collected using the LCP injector and compared with the
structure of PC obtained previously using the GDVN injector.
Both structures were of very similar resolution and quality.
During the LCP–SFX experiment, data were collected from
less than 7 ml of LCP sample (containing 3 ml crystal suspension), which yielded 18 794 hits, from which 6629 patterns
were indexed at 1.75 Å resolution (Table 2). The quality of the
structural model can be assessed from the details presented
for one of the chromophores of PC shown in Fig. 3. The
simulated-annealing composite OMIT 2mFo  DFc electrondensity map at a contour level of 1.5 fits tightly to the
chromophore, which is difficult to achieve (even at higher

Figure 3
Phycocyanin structure. Simulated-annealing composite OMIT
2mFo  DFc electron-density map contoured at the 1.5 level for the
chromophore phycobilin inside its binding pocket. The map was built
using 1.75 Å resolution XFEL data from PC crystals delivered in LCP.
IUCrJ (2015). 2, 545–551

resolution). Most importantly, using the LCP–SFX technique,
the amount of protein crystals used is dramatically reduced,
even though crystallization was carried out in liquid medium
(as described in x2). Hence, previously established crystallization conditions can be successfully coupled with this
technique. The entire data set required just 3 ml of crystal
suspension (0.1 mg of protein), compared with the hundreds
of microlitres of crystal suspension (30 mg of protein) that
were used for the GDVN structure. The quality of the
resulting electron-density map is very high, despite only 6629
crystals contributing to the data set.

4. Discussion
Several considerations affect the use of LCP as a delivery
matrix, the most important of which are the choice of the LCP
host lipids and the optimal crystal size and density. The lipid
choice is dictated by two factors. The first factor is the
compatibility of the lipids with LCP extrusion in vacuum. The
XFEL beam path usually runs in vacuum to reduce X-ray
scattering. Previously, we have observed that the most
commonly used lipid for LCP crystallization, monoolein, can
partially solidify, forming the lamellar crystalline Lc phase.
upon the injection of monoolein-based LCP in vacuum
(Weierstall et al., 2014). The Lc phase produces strong
diffraction that can damage the detector; therefore, it should
be avoided. Monoolein belongs to a lipid class known as
monoacylglycerols (MAGs), which are composed of glycerol
attached to a single fatty-acid chain through an ester bond.
Monounsaturated MAGs used commonly for LCP applications are often referred to as N.T MAG based on the number
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of carbon atoms between the ester and the double bond (N)
and between the double bond and the terminal methyl group
(T). Therefore, monoolein corresponds to 9.9 MAG in this
terminology. We have found that the shorter-chained monoolein analogues 9.7 MAG and 7.9 MAG, as well as a 1:1
mixture of 9.9 MAG and 7.9 MAG, do not form the Lc phase
upon injection into vacuum and therefore are suitable as host
lipids when LCP is extruded in vacuum. We should note that
this limitation is relieved when LCP is extruded at atmospheric pressure, and all common LCP-forming lipids that were
tested, including monoolein, can be used for this purpose.
The second factor is the compatibility of LCP with the
precipitant solution used for protein crystallization. Many
components of crystallization cocktails can disrupt LCP when
used at high concentrations or extreme pH values (Cherezov
et al., 2001; Joseph et al., 2011). Of all of the MAGs, monooleinbased LCP is one of the most stable towards the broadest
range of commonly used precipitants. It is likely that new
LCP-forming lipids with even higher stability will be identified. The compatibility of the chosen precipitant solution
should be tested with different combinations of lipids before
producing the actual samples containing protein crystals.
Instability of LCP towards a particular precipitant composition can often be rectified by lowering the concentration of
one of the components or by replacing one of the components
by another, if this does not adversely affect the stability of the
protein crystals. In the case of lysozyme, the precipitant was
compatible with 7.9 MAG, 9.7 MAG and 9.9 MAG/7.9 MAG
mixture lipids, while the PC precipitant was not compatible
with 9.7 MAG but worked well with a 1:1 9.9 MAG:7.9 MAG
mixture.
Data collected by SFX contain many randomly changing
parameters, such as the exact crystal orientation, the crystal
size and mosaicity and the XFEL pulse intensity and energy.
Data processing relies on averaging of all these fluctuations,
in the simplest case by Monte Carlo integration over many
observations of the same reflection, and this requires a high
multiplicity of data for convergence. Therefore, for efficient
SFX data collection it is important to attain a high density of
microcrystals, ensuring a high crystal hit rate. A clear advantage of working with crystals of soluble proteins, compared
with membrane-protein crystals grown in LCP, is the ability
to concentrate the crystal slurry to the desired concentration
simply by centrifugation. Thus, the optimal concentration of
crystals in LCP can be achieved, which corresponds to a 30–
40% hit rate (number of images with crystal diffraction/total
number of images). Higher hit rates would increase the
occurrence of diffraction patterns from multiple crystals owing
to the high density of crystals in the LCP stream, which is
undesirable. Having adjusted the crystal density to an optimal
value, we could collect full data sets in the shortest time:
43 min for lysozyme and 40 min for PC.
While this manuscript was under preparation, a paper
describing soluble protein crystal delivery in a grease matrix
using a different injector was published (Sugahara et al., 2014).
One of the principal differences between grease and LCP as
the crystal-delivery matrix is that in LCP the crystals remain

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in contact with the original precipitant solution, while upon
mixing with grease most of the precipitant solution is
removed. We anticipate that different crystals could have
different stabilities in such disparate matrices, and therefore
further development of both of these and other approaches
will expand the usage of SFX for challenging soluble protein
targets at XFEL sources.

Acknowledgements
This work was supported by NIH/NIGMS grant R01
GM108635 (VC), the Center for Applied Structural Discovery
(CASD) at the Biodesign Institute at ASU (RF, WL, SB, SR
UW, JCHS and PF), NSF Science and Technology Center
grant 1231306 (JCHS, PF, UW and VC), the Helmholtz
Association, the German Research Foundation (DFG)
Cluster of Excellence ‘Center for Ultrafast Imaging’ and the
German Federal Ministry of Education and Research (BMBF)
project FKZ 05K12CH1 (AB, TAW and MM). MM acknowledges support from the Marie Curie Initial Training Network
NanoMem (grant 317079). Use of the Linac Coherent Light
Source (LCLS), SLAC National Accelerator Laboratory is
supported by the US Department of Energy, Office of Science,
Office of Basic Energy Sciences under Contract No.
DE-AC02-76SF00515. We thank Cornelius Gati, Marc
Messerschmidt, Mark Hunter and Nadia Zatsepin for help
with data collection and processing.

References
Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J.,
Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev,
A., Zwart, P. H. & Adams, P. D. (2012). Acta Cryst. D68, 352–367.
Barty, A., Kirian, R. A., Maia, F. R. N. C., Hantke, M., Yoon, C. H.,
White, T. A. & Chapman, H. (2014). J. Appl. Cryst. 47, 1118–1131.
Boutet, S. & Williams, G. J. (2010). New J. Phys. 12, 035024.
Boutet, S. et al. (2012). Science, 337, 362–364.
Caffrey, M. & Cherezov, V. (2009). Nat. Protoc. 4, 706–731.
Chapman, H. N. et al. (2006). Nat. Phys. 2, 839–843.
Chapman, H. N. et al. (2011). Nature (London), 470, 73–77.
Cheng, A., Hummel, B., Qiu, H. & Caffrey, M. (1998). Chem. Phys.
Lipids, 95, 11–21.
Cherezov, V., Fersi, H. & Caffrey, M. (2001). Biophys. J. 81, 225–242.
DePonte, D. P., Weierstall, U., Schmidt, K., Warner, J., Starodub, D.,
Spence, J. C. H. & Doak, R. B. (2008). J. Phys. D Appl. Phys. 41,
195505.
Duisenberg, A. J. M. (1992). J. Appl. Cryst. 25, 92–96.
Feld, G. K. & Frank, M. (2014). Curr. Opin. Struct. Biol. 27, 69–78.
Garman, E. F. (2014). Science, 343, 1102–1108.
Hart, P. et al. (2012). Proc. SPIE, 8504, 85040C.
Johansson, L. C. et al. (2013). Nat. Commun. 4, 2911.
Johansson, L. C. et al. (2012). Nat. Methods, 9, 263–265.
Joseph, J. S., Liu, W., Kunken, J., Weiss, T. M., Tsuruta, H. &
Cherezov, V. (2011). Methods, 55, 342–349.
Kabsch, W. (2010). Acta Cryst. D66, 133–144.
Karplus, P. A. & Diederichs, K. (2012). Science, 336, 1030–1033.
Kern, J. et al. (2013). Science, 340, 491–495.
Kirian, R. A., White, T. A., Holton, J. M., Chapman, H. N., Fromme,
P., Barty, A., Lomb, L., Aquila, A., Maia, F. R. N. C., Martin, A. V.,
Fromme, R., Wang, X., Hunter, M. S., Schmidt, K. E. & Spence,
J. C. H. (2011). Acta Cryst. A67, 131–140.
IUCrJ (2015). 2, 545–551

research papers
Kupitz, C., Grotjohann, I., Conrad, C. E., Roy-Chowdhury, S.,
Fromme, R. & Fromme, P. (2014). Philos. Trans. R. Soc. B Biol. Sci.
369, 20130316.
Leslie, A. G. W. (2006). Acta Cryst. D62, 48–57.
Liu, W., Ishchenko, A. & Cherezov, V. (2014). Nat. Protoc. 9, 2123–
2134.
Liu, W. et al. (2013). Science, 342, 1521–1524.
Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. & Hajdu, J.
(2000). Nature (London), 406, 752–757.

IUCrJ (2015). 2, 545–551

Paithankar, K. S., Owen, R. L. & Garman, E. F. (2009). J. Synchrotron
Rad. 16, 152–162.
Redecke, L. et al. (2013). Science, 339, 227–230.
Sawaya, M. R. et al. (2014). Proc. Natl Acad. Sci. USA, 111, 12769–
12774.
Sugahara, M. et al. (2014). Nat. Methods, 12, 61–63.
Weierstall, U. et al. (2014). Nat. Commun. 5, 3309.
White, T. A., Kirian, R. A., Martin, A. V., Aquila, A., Nass, K., Barty,
A. & Chapman, H. N. (2012). J. Appl. Cryst. 45, 335–341.

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SFX of proteins in LCP

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