Viscous hydrophilic injection matrices for serial crystallography

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates – gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used forin mesomembrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydrogels as viscous injection matrices is described, namely sodium carboxymethyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements.

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High-viscosity injector-based pink-beam serial crystallography of microcrystals at a synchrotron radiation source

IUCrJ ◽

10.1107/s205225251900263x ◽

2019 ◽

Vol 6 (3) ◽

pp. 412-425 ◽

Cited By ~ 17

Author(s):

Jose M. Martin-Garcia ◽

Lan Zhu ◽

Derek Mendez ◽

Ming-Yue Lee ◽

Eugene Chun ◽

...

Keyword(s):

Synchrotron Radiation ◽

Radiation Source ◽

Structural Changes ◽

High Viscosity ◽

Synchrotron Radiation Source ◽

Proteinase K ◽

Moderate Increase ◽

Time Resolved ◽

X Ray ◽

Serial Crystallography

Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth (`pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A2A adenosine receptor (A2AAR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A2AAR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.

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Lard Injection Matrix for Serial Crystallography

International Journal of Molecular Sciences ◽

10.3390/ijms21175977 ◽

2020 ◽

Vol 21 (17) ◽

pp. 5977

Author(s):

Ki Hyun Nam

Keyword(s):

Flow Rate ◽

Room Temperature ◽

Glucose Isomerase ◽

Viscous Medium ◽

Cubic Phase ◽

Time Resolved ◽

Crystal Sample ◽

X Ray ◽

Serial Crystallography ◽

Delivery Medium

Serial crystallography (SX) using X-ray free electron laser or synchrotron X-ray allows for the determination of structures, at room temperature, with reduced radiation damage. Moreover, it allows for the study of structural dynamics of macromolecules using a time-resolved pump-probe, as well as mix-and-inject experiments. Delivering a crystal sample using a viscous medium decreases sample consumption by lowering the flow rate while being extruded from the injector or syringe as compared to a liquid jet injector. Since the environment of crystal samples varies, continuous development of the delivery medium is important for extended SX applications. Herein, I report the preparation and characterization of a lard-based sample delivery medium for SX. This material was obtained using heat treatment, and then the soluble impurities were removed through phase separation. The lard injection medium was highly stable and could be injected via a syringe needle extruded at room temperature with a flow rate < 200 nL/min. Serial millisecond crystallography experiments were performed using lard, and the room temperature structures of lysozyme and glucose isomerase embedded in lard at 1.75 and 1.80 Å, respectively, were determined. The lard medium showed X-ray background scattering similar or relatively lower than shortenings and lipidic cubic phase; therefore, it can be used as sample delivery medium in SX experiments.

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A fully automated crystallization apparatus for small protein quantities

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20015514 ◽

2021 ◽

Vol 77 (1) ◽

Author(s):

Ryuichi Kato ◽

Masahiko Hiraki ◽

Yusuke Yamada ◽

Mikio Tanabe ◽

Toshiya Senda

Keyword(s):

Protein Crystallization ◽

Diffusion Method ◽

Cubic Phase ◽

Web Browser ◽

X Ray ◽

X Ray Crystallography ◽

Membrane Protein Crystallization ◽

Crystallization Conditions ◽

Set Up ◽

Very High

In 2003, a fully automated protein crystallization and monitoring system (PXS) was developed to support the structural genomics projects that were initiated in the early 2000s. In PXS, crystallization plates were automatically set up using the vapor-diffusion method, transferred to incubators and automatically observed according to a pre-set schedule. The captured images of each crystallization drop could be monitored through the internet using a web browser. While the screening throughput of PXS was very high, the demands of users have gradually changed over the ensuing years. To study difficult proteins, it has become important to screen crystallization conditions using small amounts of proteins. Moreover, membrane proteins have become one of the main targets for X-ray crystallography. Therefore, to meet the evolving demands of users, PXS was upgraded to PXS2. In PXS2, the minimum volume of the dispenser is reduced to 0.1 µl to minimize the amount of sample, and the resolution of the captured images is increased to five million pixels in order to observe small crystallization drops in detail. In addition to the 20°C incubators, a 4°C incubator was installed in PXS2 because crystallization results may vary with temperature. To support membrane-protein crystallization, PXS2 includes a procedure for the bicelle method. In addition, the system supports a lipidic cubic phase (LCP) method that uses a film sandwich plate and that was specifically designed for PXS2. These improvements expand the applicability of PXS2, reducing the bottleneck of X-ray protein crystallography.

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Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319012695 ◽

2019 ◽

Vol 75 (10) ◽

pp. 937-946 ◽

Cited By ~ 3

Author(s):

Rebecka Andersson ◽

Cecilia Safari ◽

Petra Båth ◽

Robert Bosman ◽

Anastasya Shilova ◽

...

Keyword(s):

Membrane Proteins ◽

Protein Structures ◽

Cubic Phase ◽

Crystal Formation ◽

X Ray Diffraction ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography ◽

Lipidic Cubic Phase ◽

Serial Crystallography

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

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A Droplet-Based, Composite PDMS/Glass Capillary Microfluidic System for Evaluating Protein Crystallization Conditions by Microbatch and Vapor-Diffusion Methods with On-Chip X-Ray Diffraction

Angewandte Chemie International Edition ◽

10.1002/anie.200453974 ◽

2004 ◽

Vol 43 (19) ◽

pp. 2508-2511 ◽

Cited By ~ 259

Author(s):

Bo Zheng ◽

Joshua D. Tice ◽

L. Spencer Roach ◽

Rustem F. Ismagilov

Keyword(s):

Protein Crystallization ◽

Microfluidic System ◽

X Ray Diffraction ◽

Glass Capillary ◽

Vapor Diffusion ◽

X Ray ◽

Crystallization Conditions ◽

On Chip

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Approach of Serial Crystallography

Crystals ◽

10.3390/cryst10100854 ◽

2020 ◽

Vol 10 (10) ◽

pp. 854

Author(s):

Ki Hyun Nam

Keyword(s):

Radiation Damage ◽

Room Temperature ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography ◽

The Past ◽

Wide Range ◽

Experimental Limitation ◽

Serial Crystallography ◽

Collection Strategies

Radiation damage and cryogenic sample environment are an experimental limitation observed in the traditional X-ray crystallography technique. However, the serial crystallography (SX) technique not only helps to determine structures at room temperature with minimal radiation damage, but it is also a useful tool for profound understanding of macromolecules. Moreover, it is a new tool for time-resolved studies. Over the past 10 years, various sample delivery techniques and data collection strategies have been developed in the SX field. It also has a wide range of applications in instruments ranging from the X-ray free electron laser (XFEL) facility to synchrotrons. The importance of the various approaches in terms of the experimental techniques and a brief review of the research carried out in the field of SX has been highlighted in this editorial.

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Isoprenoid-chained lipid EROCOC17+4: a new matrix for membrane protein crystallization and a crystal delivery medium in serial femtosecond crystallography

Scientific Reports ◽

10.1038/s41598-020-76277-x ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Kentaro Ihara ◽

Masakatsu Hato ◽

Takanori Nakane ◽

Keitaro Yamashita ◽

Tomomi Kimura-Someya ◽

...

Keyword(s):

Protein Crystallization ◽

Water System ◽

Cubic Phase ◽

Marked Contrast ◽

X Ray ◽

X Ray Scattering ◽

Membrane Protein Crystallization ◽

Almost All ◽

Serial Femtosecond Crystallography ◽

Delivery Medium

Abstract In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method.

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