J. Biol. Macromol., 18(2), 15-22 (2018)

Review

Microcrystal-carrier matrices for serial crystallography
Michihiro Sugahara1,2*, Takanori Nakane3,4, Eriko Nango1,2, Kensuke Tono5, Makina Yabashi1 andSo Iwata1,2
1

RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho,
Sakyo-ku, Kyoto 606-8501, Japan
3Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
4(Present Address) MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis
Crick Avenue, Cambridge CB2 OQH, UK
5Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198,
Japan,
2

Received May 18, 2018
Serial femtosecond crystallography (SFX) with femtosecond X-ray pulses from X-ray freeelectron lasers (XFELs) offers a route to overcome radiation damage to small protein crystals
via the 'diffraction-before-destruction' approach. A single-pulse X-ray exposure completely
destroys individual crystals; therefore, fresh specimens must be supplied for subsequent X-ray
pulses to continue data acquisition. A matrix method using protein crystal carriers, such as
grease and oil-free hydrogel media, has been developed for serial sample loading. The microextrusion technique employing matrices allows the structure determination of a wide variety ofproteins with low sample consumption, typically less than 1 mg. This offers new opportunities
for time-resolved studies of light-driven structural changes and chemical dynamics using pumpprobe techniques at XFEL and synchrotron facilities.
Key words: X-ray free-electron laser, serial femtosecond crystallography, protein crystal, viscous
carrier media, grease matrix
Serial crystallography at XFEL andbiomolecules,

other facilities

recognition, catalysis and regulation. Thisfor

example,substrate

High resolution structures provide key

information can then be used to control theirinsights into functions and mechanisms offunctions by drugs or exploit them as tools in*To whom correspondence should be
addressed: msuga@spring8.or.jp

biologyandchemistry.

Highbrilliancesynchrotron-based X-ray sources, sensitive two-16

facility is designed to operate up to ﬁvebeamlines (BLs) with independent undulators.
As of 2018, three BLs can be operated
simultaneously. User operation of BL3 for hard
X-ray FEL and BL1 for wide-range spontaneousradiation began in 2012. The BL1 has been
upgraded to produce soft X-ray FEL with adedicated linac called SCSS+. The upgraded
Fig. 1. The SPring-8 Angstrom Compact freeBL1 has been open to users since 2016. The BL2electron LAser (SACLA), RIKEN SPring-8

was constructed in 2013 as the second hard X-

Center, Hyogo, Japan.

ray FEL beamline. Parallel operation of BL2 andBL3 began in 2017.

dimensional

detectors

andcryo-coolingtechniques have facilitated high resolution
structure
Nevertheless,determinationofproteins.

specimens

analysedusing

traditional X-ray crystallography techniques
suffer from radiation-induced structural changes.
XFELs have created new opportunities instructural biology by allowing acquisition ofdiffraction patterns before the onset of X-ray
radiation damage via ultra-fast X-ray exposuresin the femtosecond (fs) region. This is known as

Serial femtosecond crystallography (SFX)(Fig. 2) has been established at the LCLS(4–7). A
single XFEL pulse completely destroys small
individual crystals, meaning that each specimen
survives only for a single shot, necessitating the
supply of fresh crystals for subsequent X-ray
pulses to continue data acquisition. SFX allows
data collection from small protein crystals on the
micrometre
determinetosubmicrometerroom-temperaturescales

SFX also enables time-resolved experiments'diffraction before destruction'(1). Several XFEL
facilities have been constructed; the Linac
Coherent Light Source (LCLS) in USA started
user operation in 2009 as the first hard X-ray
FEL. The SPring-8 Angstrom Compact freeelectron LAser (SACLA) in Japan followed in2012. PAL-XFEL in South Korea and theEuropean XFEL in Germany started in 2017,
and the SwissFEL in Switzerland is planned for
2019.
SACLA(2) (Fig. 1) provides ultrafast X-ray

Fig. 2. Serial femtosecond crystallography atpulses with a duration shorter than 10 fs at a

SACLA.

wide photon energy range from 4 to 20 keV (4–supplied for subsequent X-ray pulses.

15 keV for user operation)(3). The SACLA

tostructures(8–15).

Freshnano/microcrystals

are

17

even

with

femtosecond

time

resolution.

jet consumes 10–100 mg of protein sample, andReactions are typically triggered by an opticalthis high rate of sample consumption impedes

laser or substrate mixing(16–22). As a diffraction

structure determination of proteins whose

pattern is recorded with a single X-ray pulse,

production is often difficult.

non-cyclic, irreversible processes can be studied.

For membrane proteins, Uwe Weierstall

Data collection from smaller crystals, madeand coworkers at Arizona State University

possible by SFX, has the advantage of probing

developed a lipidic cubic phase (LCP) injector.

more

temporally

The viscosity of LCP means that it can besynchronised reactions, as smaller crystal

operated at a lower flow rate of ~0.2 μl/min(31) ,volume allows better laser permeation and fastera significant improvement on GDVN injectors.

substrate diffusion throughout the crystal. Serial

Using the same basic concept, we developed an

crystallography is also being adapted to

injector that extrudes viscous samples in LCP.

synchrotron radiation(23,24).

The injector consists of a hydraulic cylinder, aspatially

uniform,

removable sample reservoir and a nozzle, and is
Serial sample loading for serial

mounted in the DAPHNIS chamber. In order tocrystallography

inject protein crystals of a wide variety ofA gas dynamic virtual nozzle (GDVN),

soluble and membrane proteins at low flow rates,

which generates a liquid jet with a co-flowing

we have introduced mineral oil-based grease as

gas to stabilise and reduce the jet diameter, isa protein carrier (grease matrix) in SFX(14).

mainly used to inject small protein crystals in

Micro-extrusion of small protein crystals using

their mother liquor into the XFEL beam at

a variety of grease(14,32,33) achieves a stable

LCLS(25). A GDVN has also been applied tosample stream at a low flow rate of ~0.5 μl/min

mix-and-inject serial crystallography to study

or less, which reduces sample consumption to

enzyme–substrate interactions in real-time(26–29).

less than 1 mg.

In the first stage of the SFX research at SACLA,a GDVN-based liquid jet injector was installed

Highlyinto an experimental platform DAPHNIS

media

viscouscrystalcarrier(Diverse Application Platform for Hard X-ray

Mineral oil-based grease(14) gives adequate

Diffraction in SACLA), which consists of aprotection against cracking and dissolution ofsample chamber, a fluid injector and a custom-

protein crystals, which is the reason why mineralbuilt 4-megapixel detector with multi-port CCD

oil is used as a versatile cryoprotectant for

sensors(30). However, continuous flow extraction

protein crystals in X-ray crystallography(34). We

with a liquid jet injector requires a relatively

have also introduced Super Lube synthetic

high flow speed of ~10 m/sec. At lower rates, thegrease(32) and Super Lube nuclear grade

flow is disrupted by the surface tension of water,

approved grease (nuclear grease)(33), which give

resulting in dripping. Thus, a suitably fast liquid

lower background scattering than the mineral

18

oil-based grease. Although grease media havepotential as a versatile matrix carrier, even the
nuclear grease produces strong X-ray scatteringin the resolution range of 4–5 Å, hence
increasing noise levels. To improve the signalto-noise ratio of diffraction data, we haveintroduced a hydrogel matrix technique using
hyaluronicacid(32)

andhydroxyethyl

cellulose(33). We confirmed that the hydrogel
matrices give lower background scattering
compared to the scattering generated by a grease.
In the matrix technique, the first step is to find asuitable carrier medium for the protein crystals.
Weoccasionallyobservedcrackinganddissolution of soluble and membrane protein
crystals in the grease or hydrogel matrix,
respectively. Grease and hydrogel crystal
carriers are therefore complementary.
In the grease matrix method, grease andcrystals are mixed together typically in a ratio of
9:1 (v/v) (Fig. 3). For example, after a 100-μl

Fig. 3. Grease matrix method. (a)Proteinase K microcrystals used for SFX

measurements. (b) Grease and crystals
are dispensed on a glass slide and thenmixed with a spatula. (c) Sample
extrusion through a 100-μm-i.d. injector
nozzle.

sample of crystal suspension (e.g. a crystal
density of ~107 crystals/ml) (Fig. 3a) is
centrifuged, a 90-μl aliquot of supernatantsolution is removed, in order to increase thepatterns can be collected, which provides
sufficient multiplicity for high resolution
structure determination. Although higher hit

crystal density. A 10-μl aliquot of the crystal

rates can be achieved with increased crystal

solution is dispensed into 90 μl of grease on adensities, this would cause multiple crystal hits

glass slide and then mixed with a spatula (Fig.
3b). Typically, ~30 μl of protein crystals (size 5–
20 μm; crystal density ~107 crystals/ml) aredispersed in a matrix and mounted on an injector.
The flow rate is usually ~0.5 μl/min through a
100-μm-i.d. injector nozzle (Fig. 3c). Up to
100,000 images can be collected during 1 hourof data collection at 30 Hz. Assuming an index
rate of 20% to 30%, 20,000–30,000 indexed

in one XFEL shot, resulting in spot overlaps andmis-indexing. A crystal density up to ~107
crystals/ml is ideal for SFX data collection under
our experimental conditions using 75–100-μmi.d. nozzles.

19

Example applications
To date, we have demonstrated the utilityof grease matrices in SFX at SACLA using awide variety of soluble and membrane proteins:lysozyme, glucose isomerase, thaumatin, fatty
acid-binding protein type 3 and proteinase
K(14,32,33,35),

copperreductase(15),nitritephotosystem II (PSII)(20), luciferin-regenerating
enzyme(36), the photoswitchable fluorescentprotein IrisFP(37), bacteriorhodopsin(38), bacterial
phytochrome(39), the stem domain of human
POMGnT1 and galectin(40), and thermolysin(41).
The micro-extrusion technique with greasematrices is the most common injection method
for SFX at SACLA.

Fig. 4. A close-up view of proteinase K at
1.20 Å-resolution with a 2Fo–Fc electron-

Using the hydroxyethyl cellulose matrix

density map contoured at the 2.7σ level.

and 13 keV (0.95 Å wavelength) XFEL pulses
from SACLA(42), the proteinase K crystal
structure was determined at 1.20-Å resolution

derivatised(Fig. 4)(43), a resolution allowing us to visualisecellulose matrix(33). A total of 2,000 indexedeven hydrogen atoms(44).

patterns (native/derivative: 1,000/1,000) were

Although most structures in SFX have been

crystalsusinghydroxyethyl

sufficient for Pr-SIR and SIRAS phasing of

using

proteinase K. Native sulfur SAD phasing was

homologous structures, de novo phasing fromalso successful with SFX(35,46,50). Using grease

heavy atom-derivatised crystals has also beenmatrix, we demonstrated that the structure ofdemonstrated in many studies(15,33,36,38,40,45–49).

lysozyme can be determined by SAD phasing

Using grease matrix, de novo phasing wasfrom the anomalous signal of sulfur in theaccomplished(singleprotein and chlorine in the buffer(35). For native

phasedbymolecularbyreplacement

Hg-SIRAS

isomorphous

replacementwithanomalous

SAD phasing, we collected 150,000 indexed

scattering)(36),

Cu-SAD(single-wavelength

patterns to 2.2 Å resolution at 7 keV photon

anomalous diffraction)(15), I-SAD(38), Se-/Hg-

energy (1.77 Å wavelength). These results

SAD(40)established that our matrix technique isandanomalous

Gd-MADdiffraction)(49)(multi-wavelengthat

SACLA.

Additionally, we demonstrated de novo structure
determination of proteinase K from Pr-

compatible with accurate measurements of weakanomalous signals.

20

Applications

totime-resolved

studiesThe

serial sample loading have been reported inrecent years; Vaseline (petroleum jelly)(52),femtosecond

instrumental

in

are

agarose(53), sodium carboxymethyl cellulose, thetime-dependent

thermo-reversible block polymer Pluronic F-

X-ray

observingpulsesphenomena using a pump-probe technique. The

127(54)

temporal resolution of pump-probe experimentspoly(ethylene oxide) (PEO)(55). One type ofis determined by the duration of the pump as

carrier media is not always compatible with awell as the probe. In a typical scheme of opticalgiven crystal, causing cracking and dissolution

pump–X-ray probe experiments, the probe X-of crystals due to physical or chemical stress,

ray pulse follows the pump pulse after a desired

such as osmotic shock. A wide repertoire oftime delay. The time-resolved experimentcarrier media will, therefore, be a valuablerequires that pumped crystals remain in the X-

resource for a wide variety of proteins.

ray interaction region and that the probed

andNowadays,ahigh-molecular-weightsynchrotron-based

serialcrystals flow out of the region before the next X-

crystallography is also performed using a variety

ray probe pulse. Thus, optical pump–X-rayof viscous carrier media(23,24,52,54,55). Thus, theprobe experiments require a stable stream with atechnique of sample loading using viscous

constant flow rate. In the first time-resolved SFX

media has become increasingly important notstudy at SACLA, Nango et al. reported theonly in SFX but also in serial millisecond

conformational changes of bacteriorhodopsin

crystallography using synchrotron radiation.

(crystallised in LCP) at 13 time points from

Furthermore, our matrix-based approach should

nanoseconds

be applicable to non-protein targets such as

tophotoactivation(19),milliseconds

following

using nanosecond pump-organic and inorganic nano/micro crystals.

probe device for time-resolved SFX(51). Since
our matrix method achieves a stable sample flow
with low sample consumption, it is suited fortime-resolved

Free-Electron Laser Priority Strategy Programchangesof(MEXT) and partly by Platform Project forphotosystem II (PSII) induced by 2-flash

Supporting Drug Discovery and Life Science

illumination have been captured at a resolution

Research from AMED (grant JP17am0101070).

of 2.35 Å with the grease matrix technique(20).

The authors thank all the members of the

The structure of cytochrome P450 nitric oxide

SACLA-SFX project.

The

using

This work was supported by the X-raypump-probe

techniques.

studiesAcknowledgements

structural

reductase during catalytic reactions has been
determined

using

a

combinationofaphotosensitive caged-compound (caged-NO)
and the hydroxyethyl cellulose matrix(21).
A wide variety of the crystal carriers for

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Communicated by Komori Hirofumi