Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution is determined and compared with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.

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Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam

Journal of The Royal Society Interface ◽

10.1098/rsif.2009.0142.focus ◽

2009 ◽

Vol 6 (suppl_5) ◽

Cited By ~ 112

Author(s):

Vadim Cherezov ◽

Michael A. Hanson ◽

Mark T. Griffith ◽

Mark C. Hilgart ◽

Ruslan Sanishvili ◽

...

Keyword(s):

Membrane Proteins ◽

G Protein Coupled Receptors ◽

Cubic Phase ◽

High Quality ◽

Crystal Sample ◽

X Ray ◽

Synchrotron Beam ◽

Lipidic Cubic Phase ◽

Diffraction Patterns ◽

G Protein Coupled

Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 µm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.

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Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

IUCrJ ◽

10.1107/s2052252514026487 ◽

2015 ◽

Vol 2 (2) ◽

pp. 168-176 ◽

Cited By ~ 145

Author(s):

Przemyslaw Nogly ◽

Daniel James ◽

Dingjie Wang ◽

Thomas A. White ◽

Nadia Zatsepin ◽

...

Keyword(s):

Room Temperature ◽

Cubic Phase ◽

Temperature Structure ◽

Free Electron Lasers ◽

X Ray ◽

Cubic Phases ◽

Lipidic Cubic Phase ◽

Effective Delivery ◽

Serial Femtosecond Crystallography ◽

Delivery Medium

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 proton-pump 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.

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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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The new NCPSS BL19U2 beamline at the SSRF for small-angle X-ray scattering from biological macromolecules in solution

Journal of Applied Crystallography ◽

10.1107/s160057671601195x ◽

2016 ◽

Vol 49 (5) ◽

pp. 1428-1432 ◽

Cited By ~ 22

Author(s):

Na Li ◽

Xiuhong Li ◽

Yuzhu Wang ◽

Guangfeng Liu ◽

Ping Zhou ◽

...

Keyword(s):

Data Processing ◽

Small Angle ◽

Dynamic Range ◽

Silicon Crystal ◽

High Dynamic Range ◽

Biological Macromolecules ◽

Shanghai Synchrotron Radiation Facility ◽

X Ray ◽

X Ray Scattering ◽

Ray Scattering

The beamline BL19U2 is located in the Shanghai Synchrotron Radiation Facility (SSRF) and is its first beamline dedicated to biological material small-angle X-ray scattering (BioSAXS). The electrons come from an undulator which can provide high brilliance for the BL19U2 end stations. A double flat silicon crystal (111) monochromator is used in BL19U2, with a tunable monochromatic photon energy ranging from 7 to 15 keV. To meet the rapidly growing demands of crystallographers, biochemists and structural biologists, the BioSAXS beamline allows manual and automatic sample loading/unloading. A Pilatus 1M detector (Dectris) is employed for data collection, characterized by a high dynamic range and a short readout time. The highly automated data processing pipeline SASFLOW was integrated into BL19U2, with help from the BioSAXS group of the European Molecular Biology Laboratory (EMBL, Hamburg), which provides a user-friendly interface for data processing. The BL19U2 beamline was officially opened to users in March 2015. To date, feedback from users has been positive and the number of experimental proposals at BL19U2 is increasing. A description of the new BioSAXS beamline and the setup characteristics is given, together with examples of data obtained.

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High Dynamic Range in X-ray Imaging

Advances in Intelligent Systems and Computing - Information Technology in Biomedicine ◽

10.1007/978-3-319-91211-0_4 ◽

2018 ◽

pp. 39-51

Author(s):

Przemysław Skurowski ◽

Kamila Wicher

Keyword(s):

Dynamic Range ◽

High Dynamic Range ◽

X Ray ◽

X Ray Imaging ◽

High Dynamic

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Microscope detection options for colorless protein crystals grown in lipidic cubic phases

Journal of Applied Crystallography ◽

10.1107/s0021889803013724 ◽

2003 ◽

Vol 36 (5) ◽

pp. 1295-1296 ◽

Cited By ~ 5

Author(s):

Peter Nollert

Keyword(s):

Membrane Proteins ◽

Crystal Nucleation ◽

Protein Crystal ◽

Protein Crystals ◽

Cubic Phase ◽

Polarization Microscopy ◽

Cubic Phases ◽

Lipidic Cubic Phase ◽

Surface Distortions ◽

Microscopic Techniques

The use of lipidic cubic phases as crystal nucleation and growth matrices is becoming popular and has yielded crystals of soluble and membrane proteins. So far, all of the membrane proteins crystallized by this method have been colored. This feature has facilitated the detection of the often encountered microcrystals in initial screening rounds. Indeed, small colorless protein crystals have poor optical contrast as a result of the small differences in refractive index of the protein crystal and the surrounding lipidic cubic phase. While a perfect preparation of a lipidic cubic phase is transparent and optically isotropic, in a crystallization setup it frequently disguises crystals due to cracks, inclusions, surface distortions and phase boundaries. Here, several specialized microscopic techniques and illumination conditions are compared and it is found that sufficient contrast is generated by cross polarization microscopy and by Hoffman modulation contrast microscopy for the detection of colorless protein crystals.

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