Lipidic Cubic Phase Technologies for Structural Studies of Membrane Proteins

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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Femtosecond crystallography of membrane proteins in the lipidic cubic phase

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0314 ◽

2014 ◽

Vol 369 (1647) ◽

pp. 20130314 ◽

Cited By ~ 36

Author(s):

Wei Liu ◽

Daniel Wacker ◽

Chong Wang ◽

Enrique Abola ◽

Vadim Cherezov

Keyword(s):

Membrane Proteins ◽

Structure Determination ◽

Cubic Phase ◽

Technological Advances ◽

Lipidic Cubic Phase ◽

High Resolution Structure ◽

Effects Of Radiation ◽

G Protein Coupled ◽

Serial Femtosecond Crystallography

Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins.

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Serial femtosecond crystallography of membrane proteins with a lipidic cubic phase injector

Acta Crystallographica Section A Foundations of Crystallography ◽

10.1107/s0108767313098814 ◽

2013 ◽

Vol 69 (a1) ◽

pp. s140-s141

Author(s):

Uwe Weierstall ◽

Daniel James ◽

Dingjie Wang ◽

John C. H. Spence ◽

R. B. Doak ◽

...

Keyword(s):

Membrane Proteins ◽

Cubic Phase ◽

Lipidic Cubic Phase ◽

Serial Femtosecond Crystallography

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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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Observing membrane proteins via SANS during lipidic cubic phase crystallization

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s010876731809757x ◽

2018 ◽

Vol 74 (a1) ◽

pp. a242-a242

Author(s):

Thomas E. Cleveland ◽

Paul Butler

Keyword(s):

Membrane Proteins ◽

Cubic Phase ◽

Lipidic Cubic Phase

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

IUCrJ ◽

10.1107/s2052252520012701 ◽

2020 ◽

Vol 7 (6) ◽

pp. 976-984

Author(s):

Ming-Yue Lee ◽

James Geiger ◽

Andrii Ishchenko ◽

Gye Won Han ◽

Anton Barty ◽

...

Keyword(s):

Membrane Proteins ◽

Dynamic Range ◽

High Dynamic Range ◽

Cubic Phase ◽

Experimental Setup ◽

Temperature Structure ◽

Helium Atmosphere ◽

X Ray ◽

Lipidic Cubic Phase ◽

Time Required

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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