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Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.

ABSTRACT: Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-?m-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.

SUBMITTER: Weierstall U

PROVIDER: S-EPMC4061911 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.

Weierstall Uwe U James Daniel D Wang Chong C White Thomas A TA Wang Dingjie D Liu Wei W Spence John C H JC Bruce Doak R R Nelson Garrett G Fromme Petra P Fromme Raimund R Grotjohann Ingo I Kupitz Christopher C Zatsepin Nadia A NA Liu Haiguang H Basu Shibom S Wacker Daniel D Han Gye Won GW Katritch Vsevolod V Boutet Sébastien S Messerschmidt Marc M Williams Garth J GJ Koglin Jason E JE Marvin Seibert M M Klinker Markus M Gati Cornelius C Shoeman Robert L RL Barty Anton A Chapman Henry N HN Kirian Richard A RA Beyerlein Kenneth R KR Stevens Raymond C RC Li Dianfan D Shah Syed T A ST Howe Nicole N Caffrey Martin M Cherezov Vadim V

Nature communications 20140101

Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smooth ...[more]

PMID: 24525480

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Project description:Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

Dataset Information

Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.

Publications

Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.

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