research papers

IUCrJ

Lipidic cubic phase serial millisecond
crystallography using synchrotron radiation

ISSN 2052-2525

BIOLOGY j MEDICINE

Received 16 October 2014
Accepted 1 December 2014

Edited by J. L. Smith, University of Michigan,
USA
†

Keywords: lipidic cubic phases; protein crystallography; bacteriorhodopsin; XFEL
PDB references: bacteriorhodopsin using SMX,
4x31; bacteriorhodopsin using conventional
crystallography, 4x32
Supporting information: this article has
supporting information at www.iucrj.org

Przemyslaw Nogly,a Daniel James,b Dingjie Wang,b Thomas A. White,c Nadia
Zatsepin,b Anastasya Shilova,d Garrett Nelson,b Haiguang Liu,b Linda Johansson,e
Michael Heymann,c Kathrin Jaeger,a Markus Metz,c,f Cecilia Wickstrand,g Wenting
Wu,a Petra Båth,g Peter Berntsen,g Dominik Oberthuer,c,f Valerie Panneels,a Vadim
Cherezov,e Henry Chapman,c,h Gebhard Schertler,a,i Richard Neutze,g John
Spence,b Isabel Moraes,j,k,l Manfred Burghammer,d,m* Joerg Standfussa* and Uwe
Weierstallb*
a
Laboratory for Biomolecular Research, Paul Scherrer Institute, Villigen 5232, Switzerland, bDepartment of Physics,
Arizona State University, Tempe, AZ 85287, USA, cCenter for Free-Electron Laser Science, Deutsches ElektronenSynchrotron DESY, Hamburg 22607, Germany, dEuropean Synchrotron Radiation Facility, Grenoble Cedex 9, F-38043,
France, eDepartment of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, California
USA, fCentre for Ultrafast Imaging, Hamburg 22607, Germany, gDepartment of Chemistry and Molecular Biology,
University of Gothenburg, Gothenburg, Sweden, hDepartment of Physics, University of Hamburg, Hamburg 22607,
Germany, iDeparment of Biology, ETH Zurich, Zürich 8093, Switzerland, jMembrane Protein Laboratory, Diamond Light
Source, Harwell Science and Innovation Campus, Chilton, Oxfordshire OX11 0DE, England, kDepartment of Life
Sciences, Imperial College London, London, England, lResearch Complex at Harwell Rutherford, Appleton Laboratory,
Harwell, Didcot, Oxfordshire OX11 0FA, England, and mDepartment of Analytical Chemistry, Ghent University, Ghent
B-9000, Belgium. *Correspondence e-mail: burgham@esrf.fr, joerg.standfuss@psi.ch, weier@asu.edu

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the
crystallization of membrane proteins. Moreover, the viscous LCP also provides a
highly effective delivery medium for serial femtosecond crystallography (SFX)
at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology
to perform serial millisecond crystallography (SMX) at more widely available
synchrotron microfocus beamlines is described. Compared with conventional
microcrystallography, LCP-SMX eliminates the need for difficult handling of
individual crystals and allows for data collection at room temperature. The
technology is demonstrated by solving a structure of the light-driven protonpump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature
structure of bR is very similar to previous cryogenic structures but shows small
yet distinct differences in the retinal ligand and proton-transfer pathway.

1. Introduction
Structure determination by X-ray crystallography has developed continuously over the last century, yielding structures of
ever more difficult and complex molecules. An important
development is synchrotron-based microcrystallography,
which uses brilliant X-ray beams of a few micrometres in
diameter to collect data from very small weakly diffracting
crystals. Microcrystallography has matured over the last few
years (Smith et al., 2012), but structure determination using
microcrystals remains challenging and radiation damage limits
the achievable resolution for well ordered small crystals
(Garman, 2010a). Microcrystallography has been particularly
successful with membrane proteins grown in lipidic cubic
phases (LCP). Crystallization in LCP environments often
produces crystals that are highly ordered but limited in size.
Protein crystallization in LCP was introduced 18 years ago

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doi:10.1107/S2052252514026487

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(Landau & Rosenbusch, 1996) and has proven crucial for
determining high-resolution structures and functional
mechanisms of membrane proteins from several families, such
as microbial rhodopsins, G protein-coupled receptors, ion
channels, transporters and enzymes (Cherezov, 2011).
Protein microcrystals grown in LCP are well suited for the
emerging technique of serial femtosecond crystallography
(SFX) at X-ray free-electron lasers (XFELs) (Chapman et al.,
2011; Fromme & Spence, 2011; Spence et al., 2012), in which
micro- and nanometre-sized protein crystals are injected
across ultrafast X-ray pulses in a stream at room temperature.
Due to the high flux density, each crystal is destroyed by the
photoelectron cascade following the X-ray pulse, but the
duration of each XFEL pulse is so brief (typically 40 fs) that
it terminates before conventional types of radiation damage
have manifested themselves (Neutze et al., 2000). Therefore,
only a single diffraction pattern per crystal, which contains
information on essentially undamaged molecules, is collected.
A gas dynamic virtual nozzle (GDVN) (DePonte et al., 2008;
Weierstall et al., 2012), which was the injection device for these
first experiments (Chapman et al., 2011; Boutet et al., 2012;
Johansson et al., 2012), can deliver crystals in their lowviscosity crystallization buffer/mother liquor at a liquid flow
rate of about 10 ml min1 and a speed of about 10 m s1. At
this flow rate, and with the repetition rate of the hard XFEL
sources currently in operation, most crystals flow past the
interaction point in the time between X-ray pulses and are
therefore wasted. This results in a requirement of up to 100 mg
of protein for a single complete data set, and obtaining such
large amounts is not feasible for many membrane proteins.
Due to its high viscosity, the LCP can be extruded at much
lower stream speeds (1–300 nl min1), but it is incompatible
with the GDVN device. A newly developed LCP injector
(Weierstall et al., 2014) extrudes a 20–50 mm diameter stream
of LCP into ambient air or vacuum. It reduces sample
consumption 50–100 fold compared with the GDVN and has
already been used to solve several membrane protein structures (Liu et al., 2013; Caffrey et al., 2014; Weierstall et al.,
2014) at the Linac Coherent Light Source (LCLS, Stanford,
California, USA), the first hard X-ray FEL.
Recently, the structural biology community has begun to
adopt serial approaches to structure determination at thirdgeneration synchrotron sources. Gati et al. (2014) used helical
line scans to solve the structure of Trypanosoma brucei
procathepsin B from cryocooled crystals. The first serial
crystallography experiment at a synchrotron yielded a 2.1 Å
lysozyme structure by merging single frames from microcrystals injected randomly into the X-ray beam in a glass
capillary (Stellato et al., 2014). Our LCP windowless injector
allows the stream velocity to be slowed down to a rate of 0.05–
0.15 mm ms1, which allows 10–100 ms exposure times and
efficient use of the sample. Here, we demonstrate that this
makes it possible to perform LCP microjet-based serial
millisecond crystallography (SMX) using synchrotron radiation, similar to SFX at an XFEL. The key advantages of this
method are: (i) crystal injection using the LCP combined with
a microfocus beamline allows diffraction data to be collected
IUCrJ (2015). 2, 168–176

at room temperature, and hence crystal freezing and difficult
crystal handling steps such as mounting crystals in a loop are
not necessary; (ii) thousands of crystals can be screened in a
short time and with less than a milligram of protein; (iii)
microfocus beams at storage-ring sources are widely available
and hence beam access is unlikely to limit SMX; and (iv) the
method is well suited for time-resolved diffraction studies on
the microsecond to millisecond timescale.

2. Methods
2.1. Purification

Bacteriorhodopsin (bR) was purified from purple
membranes of Halobacterium salinarum as described by
Nollert (2004), with modifications. All steps were performed
under dim red light or in the dark. The purple membrane was
resuspended in 50 mM NaH2PO4 pH 6.9 (GERBU Biotechnik
GmbH) and 1.7% of n-octyl--d-glucopyranoside (-OG;
Anagrade, Affymetrix) was added, yielding a final bR
concentration of about 0.9 mg ml1 (as judged spectrophotometrically
at
560 nm,
absorption
coefficient
63 000 l mol1 cm1). The suspension was sonicated in a bath
sonicator for 1 min and incubated on a rock-roller. The next
day, the pH was adjusted to 5.5 with 0.1 M HCl. The insoluble
fraction was pelleted at 55 000 r min1 (Ti 70 rotor; 15 C) for
45 min and the supernatant was concentrated in Amicon Ultra
Centrifugal Filters (Ultracel-50k). The concentrated supernatant was applied onto a TSK G3000SW gel filtration column
(TOSOH Bioscience) equilibrated with 1.2% -OG in 25 mM
NaH2PO4 pH 5.5. The bR from the first peak was discarded.
The bR from the second peak was concentrated in Amicon
Ultra Centrifugal Filters (Ultracel-50k) to a final concentration of 9 mg ml1.
2.2. Crystallization

The lipidic cubic phase (LCP) was prepared by mixing the
protein sample with monoolein (Nu-Check) in a 40:60 volume
ratio using Hamilton syringes and a syringe coupler (Caffrey
& Cherezov, 2009). Up to 20 ml of the LCP was injected into a
100 ml Hamilton syringe filled with precipitant solution
composed of 29–38% polyethylene glycol 2000 (Fluka
Analytical) and 100 mM Sorensen phosphate buffer pH 5.6
(KH2PO4 and Na2HPO4 from GERBU). A shower of small
crystals appeared within a few days, with sizes varying with the
batch of purified protein and the concentration of precipitant.
All crystallization setups were prepared under dim red light
and incubated at 20 C in the dark.
2.3. Sample preparation for LCP jet

Before loading the samples into the LCP injector, the
precipitant solution was removed from the syringe and
monoolein was added. To obtain a homogenous suspension of
crystals in the LCP, samples were mixed through the syringe
coupler. During this mixing step, larger crystals were broken
into smaller fragments of less than 50 mm. The crystallization
solutions were filtered and the protein samples were
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centrifuged to minimize the presence of dust particles in the
final sample, as dust particles may block the LCP injector
nozzle (the largest diameter used was 50 mm).

2.4. Data collection and processing

Serial crystallographic data were collected at the ESRF
Microfocus Beamline (ID13; Grenoble, France) in the setup
described in the Results section. Alignment of the X-ray beam
onto the LCP stream was facilitated with a grid scan of the

area around the tip of the injector. Diffraction patterns were
collected from randomly oriented crystals with 10–50 ms
exposure times at a rate of 10–17 Hz. Due to random failures
in the Rayonix MX-170 CCD detector, the upper left quadrant
was excluded during data analysis. The detector was set to 4  4
binning mode with a pixel size of 177 mm and a frame size of
960  960 pixels. Because of overheads due to saving data on
the ESRF data server, the frame rate was limited to
17 frames s1. Collected images (details in Fig. 1) were preprocessed using Cheetah (Barty et al., 2014) to exclude images
without diffraction patterns. Higher hit rates and resolution
were observed for crystals sized 20–40 mm. The CrystFEL
program suite (White et al., 2012) was used for data processing.
The conventional Cryo data set was collected at the PXI
beamline of the Swiss Light Source (Paul Scherrer Institute,
Villigen, Switzerland) in a cryostream at 100 K (details in
Table. 1). The diffraction images were processed and scaled
using XDS (Kabsch, 2010), followed by merging using Aimless
(Evans & Murshudov, 2013).

2.5. Model building and refinement

Figure 1
Data collection and refinement statistics for the SMX and CRYO bR
structures. The upper inset shows the development of the correlation
between the two indexing possibilities over the number of crystals used to
resolve the indexing ambiguity. The middle inset shows zone-axis plots of
the data before and after solving indexing ambiguity. Colours are
proportional to the square root of the intensity (i.e. I1/2). The lower inset
plots the signal-to-noise ratio, expressed as I/(I), against the resolution
of the SMX data.

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LCP millisecond crystallography

A single molecular replacement solution was found using
the SMX data set and the structure of sensory rhodopsin II
[PDB code 1h68 (Royant et al., 2001), without ligands] as a
search model in Phaser (McCoy et al., 2007). Initial phases
were used to rebuild the bacteriorhodopsin model automatically with Phenix.autobuild (Adams et al., 2002), using
simulated annealing refinement between iterative building
steps. Manual building in Coot (Emsley & Cowtan, 2004) was
used to complete the autobuild model, except for residues 1–4
and 234–249, and four side-chains for residues Q75, S158,
K172 and R227. Further refinement was carried out using
PDBREDO (Joosten et al., 2012), which suggested one TLS
group for the whole protein chain. In a final round of model
building and refinement in Refmac5 (Murshudov et al., 2011),
the model was completed with ten water molecules, five lipid
fragments and all-trans retinal. The retinal was refined with
restrained geometry of the Schiff base at the covalent link to
Lys216.
The conventional Cryo data set was phased with Phaser,
using bacteriorhodpsin [without ligands, PDB code 2ntu
(Lanyi & Schobert, 2007)] as a search model. A single solution
was found and the model was completed and refined using
Refmac5. Retinal, 30 water molecules and eight lipid fragments were included in the last rounds of refinement. The
unresolved region included the first four and last 17 residues,
similar to the SMX structure. Moreover, a loop consisting of
residues 157–163 was not included in the model obtained with
the Cryo data, while for the SMX data it could be modelled
into a weak electron density. Poorly resolved side-chains of
K30, R164 and K172 were also not included in the model. The
statistics are listed in Table. 1. The protein structures determined using the SMX and Cryo data sets have been deposited
in the PDB with codes 4x31 and 4x32, respectively.
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Table 1
Data-collection and refinement statistics for SMX bR and cryo bR structures..

Data collection
X-ray source
Detector
Temperature (K)
Wavelength (Å)
Beam size (mm)
Average crystal size (mm)
Flux (photons s1)
Space group
Unit-cell parameters (Å,  )
Oscillation ( )/exposure (ms)
No. of collected images
No. of hits/indexed images
Total/unique reflections
Resolution range (Å)
Completeness (%)
Multiplicity
hI/(I)i
CC*†
Rsplit‡ (SMX) or Rp.i.m. (cryo) (%)
Matthews coefficient VM (Å3 Da1)
Solvent content (%)
B factor from Wilson plot (Å2)
Refinement
Resolution range (Å)
No. of reflections (total/test set)
Rwork/Rfree (%)
No. of atoms
Overall
Protein
Retinal
Water
Lipids and other
Average B factors (Å2)
Overall
Protein
Retinal
Water
Lipids and other
R.m.s. deviations
Bond lengths (Å)
Bond angles ( )
Ramachandran favoured (%)
Ramachandran outliers (%)
† CC* = [2CC1/2/(1 + CC1/2)]1/2.

‡ Rsplit = ð1=21=2 Þ

SMX

Cryo

ID13, ESRF
Rayonix MX-170 CCD
294
0.954
23
5–40  5–40  1–5
9.1  1011
P63
a = b = 62.8, c = 109.7,
 =  = 90,  = 120
n.a./10–50 (81% 25)
1343092
12982/5691
1223766/9655
36.56–2.40 (2.46–2.40)
100.0 (100.0)
127 (88.8)
3.57 (1.16)
0.981 (0.658)
22.4 (107)
2.50
50.76
45.2

PXI-X06SA, SLS
PILATUS 6M
100
1.000
50  10
50  50  10
5.9 1011
P63
a = b = 60.5, c =101.5,
 =  = 90,  = 120
0.1/150
2532
2532/2532
234541/16643
46.57–1.90 (1.94–1.90)
100.0 (100.0)
14.1 (14.3)
17.90 (1.80)
1.000 (0.841)
2.6 (50)
2.21
44.27
33.4

31.40–2.40 (2.46–2.40)
9192/441
20.5/24.9

52.42–1.90 (1.95–1.90)
15773/841
17.1/21.4

1848
1756
20
10
62

0.008
1.01
98.2
0.4
hkl

jIeven  Iodd j= 12

P

hkl

jIeven þ Iodd j.

3. Results
3.1. Experimental setup

The LCP injector was installed horizontally at a 90 angle
with respect to the X-ray beam (Fig. 2a). A constant LCP flow
of 20–60 nl min1 through a 50 mm nozzle was stabilized by a
co-axial flow of helium gas supplied at 7–10 bar (1 bar =
100 000 Pa). A video camera was used to monitor extrusion of
the LCP column (Fig. 2b). Several diffraction images from
raster scans were used to align the 2  3 mm Gaussian-shaped
X-ray beam onto the centre of the 50 mm wide LCP column,
approximately 40 mm from the end of the injector nozzle.
During data collection, a mechanical shutter interrupted the
X-ray beam to collect diffraction images with exposure times
of 10–50 ms (81% of images were collected at 25 ms) at a flux
of up to 9.1  1011 photons s1. The dead time between
IUCrJ (2015). 2, 168–176

3.2. Sample preparation and LCP
injection

Bacteriorhodopsin (bR) was the first
protein for which the structure was
solved (Pebay-Peyroula et al., 1997)
from crystals grown in the LCP
(Landau & Rosenbusch, 1996), using
data collected at the ID13 microfocus
28.50
beamline. Since LCP-grown bR crys27.11
tals diffract to high resolution and can
24.61
36.52
be easily visualized due to their purple
49.93
colour, bR is an ideal protein to
demonstrate LCP-SMX at synchrotron
0.009
1.21
sources. To produce a sufficient
98.9
number of bR crystals for our experi0.0
ment, we adapted previously published
crystallization conditions (Nollert,
2004) to a setup using 100 ml gas-tight
syringes (Supplementary Fig. S1),
similar to that described elsewhere (Liu, Ishchenko & Cherezov, 2014; Liu, Wacker et al., 2014). Once crystals had formed,
excess crystallization buffer was removed and the residual
buffer was absorbed by adding further monoolein. Manual
operations, including loading of the injector, took only a few
minutes. Less than 200 mg of protein was sufficient to fill the
20 ml reservoir of the LCP injector and collect data for 5–15 h,
depending on the flow rate of the jet. To maximize the
diffraction signal, the crystals should be as large as possible
but still pass through the 50 mm capillary of the LCP injector.
The concentration of crystals within the LCP also needs to be
as high as possible in order to maximize the rate at which
X-rays hit the crystals, but should optimally stay below the
level at which multiple diffraction patterns are observed on a
single diffraction image in order to simplify the analysis.
Overall, we achieved a hit rate (number of diffraction patterns
1877
1723
20
30
104

40.47
39.04
52.67
55.94
74.47

P

individual exposures was 55 ms which,
combined with overheads related to
the data-transfer rate, resulted in data
acquisition at 10–17 Hz. At the chosen
LCP flow rate, crystals moved 4–12 mm
during a single exposure, continuously
bringing fresh crystal sections or new
crystals into the beam. In contrast with
previous experiments at the LCLS, the
LCP-SMX experiment described here
is not performed in a vacuum environment, significantly reducing the
complexity and cost of the experimental setup. Furthermore, LCP
extrusion into air does not lead to a
phase change in a monoolein-based
LCP as observed in vacuum (Weierstall
et al., 2014), allowing collection of data
at ambient temperature and pressure
without the addition of special lipids.

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with >10 Bragg peaks/total number of images) of 0.5–2%,
which is somewhat lower than the hit rates of 3–8% reported
for similar experiments with different samples at the LCLS
(Liu et al., 2013; Weierstall et al., 2014). This is probably due to
the lower crystal density in our setup, as crystal size and crystal
density were negatively correlated in our crystallization
screening and we achieved the best diffraction with crystals of
20–40 mm, much larger than what would be ideal for data
collection at an XFEL. Together with the lower data acquisition rate of 10–17 Hz at the ESRF compared with 120 Hz at
the LCLS, the lower hit rate meant that the collection of this
data set took 3 d. Nevertheless, only 0.8 mg of protein in
200 ml of LCP was needed.
Occasionally, two or three consecutive hits were recorded
on some of the larger (40–50 mm) crystals. Bragg spots
appeared and disappeared within this sequence of consecutive
images, indicating that the rotational diffusion of crystals in
the LCP within the 80 ms between two exposures is larger
than their mosaic spread. To investigate this further, a
computer program was written to compare the orientations of
crystals in adjacent frames using the data stream output from
CrystFEL. For the fraction of data acquired with a 25 ms
exposure time (81% of the total frames), 1088 frames (26% of
the successfully indexed patterns) were found to be part of a
rotation series. The mean series length was 2.2 frames and
the maximum series length was 4 frames. Such a series of

consecutive diffraction patterns might be useful for indexing
and integration, as it resembles a small wedge of rotation data
similar to those typically collected in conventional crystallography. A further reduction in the LCP flow rate and an
increase in the frame rate could thus be used to collect more
images from the same crystal and increase the overall data
collection efficiency.
3.3. Data processing and map calculation

We collected 1 343 092 images, of which 12 982 were classified as hits using the Cheetah program (Barty et al., 2014),
giving an average hit rate of 1%. A large fraction of the
frames were found to exhibit artifacts in one quadrant of the
detector, and this quadrant was therefore ignored for all stages
of analysis. The unit-cell parameters were determined to be
a = b = 62.79 Å and c = 109.67 Å in space group P63 during
initial indexing of a subset of the data, consistent with the
known lattice of bR crystallized in LCP. Of the initial hits, 5691
images were successfully indexed and integrated by CrystFEL
(Version 0.5.3a+e2c7dbd5) without difficulty. However, the
space group of bR crystals is subject to an indexing ambiguity
[see White et al. (2013) for an extensive discussion], which was
resolved by CrystFEL using an algorithm related to one
recently developed for this purpose [Brehm & Diederichs
(2014); see Liu & Spence (2014) for a solution based on an
expectation maximization algorithm],
prior to merging the individual intensity
measurements for each symmetrically
unique reflection according to point
group 6/m (i.e. Friedel pairs were also
merged). The resolution limit of the
diffraction signal in the merged intensities was judged as 2.4 Å, based on
signal-to-noise ratios, CC*, visual
inspection of the density (Supplementary Fig. S2) and suggestions by the
PDB Redo web server (Joosten et al.,
2012).
For comparison against this SMX
data, we harvested a single bR crystal of
50  50  10 mm and collected data
at the Swiss Light Source under cryogenic conditions. This conventionally
collected data set (Cryo) has a resolution of 1.9 Å with no detectable signs
of twinning (as determined by
Phenix.xtriage; Adams et al., 2002). We
confirmed this finding using several
other single crystals, as all previously
Figure 2
described bR crystals grown in LCP
The experimental setup at the ID13 microfocus beamline. (a) (1) Microscope focused on the jet. (2)
show various degrees of twinning
LCP injector with (3) nozzle close to the beamstop. (b) A view of the LCP nozzle as seen through
(Wickstrand et al., 2014). It is possible
the microscope. LCP was extruded towards the left as viewed in this projection, and the X-ray beam
hits the stream at a distance of 40 mm from the end of the coned capillary. The capillary ID is 50 mm.
that this improvement in crystal quality
A co-flowing gas stream (green arrows) keeps the LCP stream straight. (c) Schematic diagram of the
is due to a change in crystallization
setup. The water used to drive the injector is shown in blue, the LCP in red and the gas in green. (d)
conditions, since we used polyethylene
An SMX diffraction pattern from a bR microcrystal, with visible Bragg spots extending out to 2.2 Å
resolution.
glycol as precipitant to avoid high

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able to include this loop in the SMX bR
structure despite the lower resolution.
Hierarchical cluster analysis (Wickstrand et al., 2014) reveals average
differences in the internal distance
matrix (Sij) of the order of 0.2 Å relative
to most published structures of the bR
ground state (Fig. 4). This difference is
primarily due to a small perturbation of
helices D, E and F away from helices A
and B relative to the structure solved
with the Cryo data. This effect potentially reflects an impact of cryocooling,
compressing the bR structure slightly,
rather than differences in data collection, since it is well known that cryocooling can introduce small structural
perturbations. Analysis of data from 30
proteins (Fraser et al., 2011), for
Figure 3
example, indicated that cooling changes
(Centre) Comparison of bR structures solved by SMX and conventional cryocrystallography
the side-chain conformations for 35% of
(Cryo). The protein backbone of the room-temperature SMX structure (purple) superimposes well
surface-exposed residues. Comparison
with the Cryo structure (blue). (Bottom) Retinal omit maps [blue (Cryo) or purple (SMX) mesh,
of serotonin receptor 2B (5-HT2B)
2Fo  Fc at 1.5; green mesh, Fo  Fc at 2.5] indicate increased flexibility in the -ionone ring. The
upper insets show a different rotamer for E194 involved in proton translocation, and indications for
crystal structures collected using
radiation damage on D38 exposed to the extramembrane environment.
microcrystallography under cryoconditions (Wacker et al., 2013) and by LCPSFX (Liu et al., 2013) revealed similar
concentrations of salts for future XFEL experiments. A
changes in surface residues. At a more detailed level, struccomparison of the two data sets is shown in Fig. 1.
tural comparison of the SMX and Cryo data sets reveals
An important consideration for every structure determined
rotamer changes in a series of amino acids on the bR surface,
by molecular replacement is the potential impact of model
but also in internal residues like Glu194 (Fig. 3, upper left
bias, which may hide differences between the model used for
panel). The ligand-binding pocket is identical in the SMX and
structure determination and the true structure. To limit the
Cryo structures (Fig. 3, lower insets). In each case, the retinal
impact of model bias, we used the related sensory protein
ligand is well resolved and, when omitted during refinement,
rhodopsin II [PDB code 1h6b (Royant et al., 2001), 30%
clear positive density emerges in the resulting difference maps,
sequence identity with bR] for molecular replacement with the
mFo  DFc. In both cases, retinal is covalently bound to
SMX data (supplementary Fig. S3) and subjected the solution
Lys216 and in the all-trans conformation, as expected for the
to automatic model building using simulated annealing
bR ground state. The extent to which the minor structural
refinement in Phenix (Adams et al., 2002). Manual addition of
differences observed here are physiologically relevant is
lipid fragments, water molecules and the retinal co-factor,
presently unclear.
combined with a final round of refinement, resulted in a bR
structure that is essentially free of model bias, providing a
5. Discussion
clear demonstration of the quality of our SMX data.
Several crystal structures have demonstrated the potential of
the LCP injector for SFX of membrane proteins using XFELs
4. Comparison of Cryo and SMX structures
(Liu et al., 2013; Caffrey et al., 2014; Weierstall et al., 2014). In
this study, we have adapted the technology for use at more
The synchrotron cryocooled bR structure was solved using
widely available synchrotron-based microfocus beamlines and
molecular replacement with ground-state bR (PDB code 2ntu;
have demonstrated that room-temperature LCP-SMX of
Lanyi & Schobert, 2007). Overall, this structure and the bR
membrane proteins is possible at synchrotron sources.
structure collected by SMX at room temperature are very
One of the problems that had to be overcome was the
similar (Fig. 3), with a C root mean-square deviation of
indexing ambiguity, with data collected from multiple crystals
0.54 Å. The distribution of crystallographic B factors is
with merohedral point groups. Even though 27 out of 65 space
comparable between the two structures, but the B factors are
groups may suffer from indexing ambiguities, it rarely causes
slightly higher in the SMX bR structure, probably because of
problems in conventional crystallography, as all indexing
the lower resolution or increased thermal motion of the
regimes are equally valid and only one has to be selected for
protein at room temperature. We observed weak electron
an individual large single crystal. Even in the case when data
density for the loop between helix E and helix F, and were thus
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Today, around 95% of protein structures are determined
from cryocooled crystals. While in our case the structural
differences between room-temperature and cryogenic data
collection were small, in some cases cryogenic cooling can
change the dynamic behaviour of proteins and may lead to
structural artifacts (Fraser et al., 2011; Keedy et al., 2014). The
reason why cryogenic data collection is still so dominant is that
cryocooling reduces radiation damage, which is the major
factor limiting the amount and quality of structural information that can be obtained from a protein crystal (Garman,
2010b). Primary radiation damage is a result of the X-ray
photoionization of atoms in the crystal or surrounding liquor,
and the subsequent rapid cascade of electron collisional
ionization that takes place in several
hundred femtoseconds, resulting in lowenergy solvated electrons and hydroxyl
radicals. Secondary damage refers to the
radiochemistry due to these radicals,
which are able to diffuse and react with
particular components such as metal
centres and disulfide bridges, and lead
to decarboxylation of aspartate and
glutamate residues (Allan et al., 2013;
Davis et al., 2013). Tertiary damage is
defined as the effect on the crystal
lattice and other mechanical consequences of the energy deposition in the
crystal.
A new method to limit radiation
damage is to outrun its consequences
and finish the measurement before the
damage causes significant loss of information at the resolution of interest
(Neutze et al., 2000). Even with an
exposure time of 100 ms, 50% of global
radiation damage can be avoided at
near room temperature by outrunning
secondary and tertiary damage effects
(Owen et al., 2012; Warkentin et al.,
2013). The effect is already exploited by
in situ diffraction methods, where the
crystals are directly exposed in crystallization plates and diffraction images
from dozens of crystals are merged to
give a complete data set (Axford et al.,
2012). In a similar approach, X-ray
semitransparent microfluidic chips have
been used to collect and merge partial
rotation series from multiple roomtemperature crystals (Khvostichenko et
Figure 4
al., 2014). At the Swiss Light Source, in
Cluster analysis of bR ground-state structures using hierarchical sorting. This analysis sorts
situ screening has been very successful
according to the average of the absolute value of the difference between two internal distance
as part of an integrated crystallization
matrices [Sjj(Å)] calculated on C atoms (Wickstrand et al., 2014). PDB codes are given for all
deposited wild-type structures of bR in its resting state. LCP bR structures are marked in purple.
and diffraction screening platform
The room-temperature SMX structure (bRSMX) and the structure of bR solved here using
(Bingel-Erlenmeyer et al., 2011). So far,
conventional data collection and cryocooling (bRcryo) are marked in red. Inset: The internal
such in situ diffraction techniques have
distance matrix for bRSMX–bRcryo shows that cryocooling compresses helices A and B slightly
not yet been developed into a routine
towards helices D, E and F.
from multiple crystals need to be merged, data sets are likely
to have a large enough overlap on which to base common
indexing. Single diffraction snapshots taken in serial crystallography provide only a set of partial reflection intensities, for
which there are two indexing possibilities in the present case
(space group P63). Direct merging of these images without
regard to intensities leads to the formation of a perfectly
twinned data set of higher symmetry (with equal contributions
from each indexing mode) that is very prone to model bias and
poorly suited for structure determination and refinement.
Here, we have provided an example of how indexing ambiguity can be resolved, and demonstrate that serial crystallography is not limited to non-merohedral space groups.

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research papers
method for data collection, mainly due to the modifications
needed at synchrotron beamlines, rotational limitations,
background from conventional crystallization plates and
radiation damage. The LCP injector used in our SMX
experiment provides a constant stream of fresh crystals, so that
each of the several thousand crystals contributing to the final
data set is exposed for only 10–50 ms. The total radiation dose
to the exposed region of a single crystal is thus only about
0.7 MGy, compared with the 28 MGy deposited during
collection of the conventional data set (calculated using
RADDOSE-3D; Zeldin et al., 2013). Per crystal, the dose thus
remains below the Henderson–Garman safe dose limit of
1 MGy at room temperature and 30 MGy for frozen samples
(Garman & McSweeney, 2007). Indeed, we could not detect
radiation damage by investigation of radiation-sensitive residues in the SMX structure, although it has been argued that
very subtle shifts near the retinal Schiff base can be observed
in high-resolution cryo structures of bR at a dose as low as
0.06 MGy (Borshchevskiy et al., 2014). By contrast, the
structure obtained from cryocooled bR crystals (28 MGy
dose) showed weak density for several aspartate residues
(Fig. 3, upper right inset), an indication of mild radiation
damage that could possibly be avoided by further truncation
of the data. Nevertheless, this comparison shows that serial
injection of crystals at a synchrotron using an LCP injector
allows collection of data at room temperature with minimal
radiation damage.
A further potential application for LCP-SMX at room
temperature is time-resolved crystallography. Cryocooled
crystals have been used extensively for low-temperature
trapping of bR intermediates [reviewed by Wickstrand et al.
(2014)] but cannot be used for genuinely time-resolved pumpprobe experiments. Room-temperature LCP-SMX can be
adapted for time-resolved diffraction studies by using laser
pulses to photoactivate the microcrystals and varying the time
of arrival of the photoactivating laser pulse relative to when
the X-ray image is recorded. By using rapid readout X-ray
detectors, it is already possible to achieve millisecond time
resolution. The use of polychromatic X-ray beams in combination with microfocusing should allow time-resolved SMX to
be extended to timescales as short as 100 ps in favourable
cases (Schotte et al., 2003). Resolving dynamics on timescales
much faster than this will remain dependent on the femtosecond pulses of XFELs (Arnlund et al., 2014).
A common problem with LCP is its high viscosity, which
makes crystal harvesting a somewhat difficult procedure,
especially for inexperienced users. In addition, crystals are
often invisible through the beamline camera, owing to the
opacity of the cryocooled lipidic mesophase surrounding
them. This makes alignment with the X-ray beam a timeconsuming process for which specific techniques like X-ray
beam rastering (Cherezov et al., 2009) or X-ray imaging
(Warren et al., 2013) are required. Serial injection of microcrystals by LCP-SMX, as demonstrated here, has the potential
to simplify this process by eliminating pre-exposure, handling
and centring of crystals. Improvements in sample preparation,
as well as the use of next-generation single-photon counting
IUCrJ (2015). 2, 168–176

X-ray pixel detectors with a higher frame rate beyond 1 kHz
frequency and a shorter readout dead time in the microsecond
range, will allow matching of the rate at which crystals traverse
the beam with the desired exposure time, resulting in more
efficient data collection. The practically zero readout noise of
modern detectors compared with the CCD detector we were
limited to in our experiment will further increase the achievable resolution. Another important factor is background
scattering from the stream of LCP. It is most prevalent in a
diffuse ring around 4.5 Å resolution and less compromising for
lower and higher resolution ranges. Data collection using
nozzles with a smaller diameter will further decrease background scattering and increase the quality of collected data,
but such nozzles have to be chosen carefully according to the
crystal size so as to not block the injector. Judicious choice of
flow rate, jet diameter, crystal size and exposure time may also
allow sufficient microcrystal rotation during an exposure to
generate fuller reflections. Future upgrades to modern
synchrotron sources will increase the available flux by several
orders of magnitude and further reduce exposure times and
the size of crystals that can be measured. With these
improvements, the method could be particularly useful for the
investigation of pharmacologically relevant human proteins
that are often expressed in only small quantities. The simplified crystal handling, compared with conventional crystal
harvesting and cryofreezing, should be well suited for automation and high-throughput approaches. We also foresee
synergies for synchrotron-based SMX and XFEL-based SFX,
as these complementary approaches are used to accelerate the
pace of discovery for the most challenging classes of proteins
in structural biology.

Acknowledgements
The authors thank Oleksandr Yefanov and Anton Barty for
discussions on the processing of serial data, and Guido Capitani and Wolfgang Brehm for remarks on how to handle the
indexing ambiguity. For the implementation of auxiliary
infrastructure at the ID13 beamline, we thank Lionel Lardière,
Michael Sztucki, Britta Weinhausen and Thomas Dane. The
Rayonix MX-170 CCD area-detector was on loan from
Theyencheri Naryanan at the ESRF ID02 beamline. We thank
Anuschka Paulhun and Vincent Olieric from the Macromolecular Crystallography group at the Swiss Light Source for
support during collection of the conventional cryogenic data
set and in situ testing of crystals. This work was financially
supported by the Swedish Research Council (PBa), the Knut
and Alice Wallenberg Foundation (PBe), NIH grant
R01GM108635 (VC), the Röntgen-Ångström-Cluster project
(05K2012) of the German Bundesministerium für Bildung und
Forschung (BMBF) (DO and HC) and NSF STC award
1231306 (DJ, DW, NZ, GN, HL and UW). The Membrane
Protein Laboratory at Diamond Light Source is funded by the
Wellcome Trust (grant No. WT089809). We further acknowledge NIH award 025979 (GN) and Swiss National Science
Foundation grants 310030_153145 (GS) and 31003A_141235
(JSt). The project was further supported by the European
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research papers
Union under the programme FP7-PEOPLE-2011-ITN
NanoMem (RN, HC, VP, IM, AS, MB, GS, MM and WW).

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