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Membrane Protein Preparation for Serial Crystallography Using High-Viscosity Injectors: Rhodopsin as an Example

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)

Abstract

Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease. They are inserted in cell membranes, are often intrinsically flexible, and can adopt several conformational states to carry out their function. Although most overall folds of membrane proteins are known, many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination. Here, using the example of rhodopsin, we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature, a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for classical single-crystal analysis even in cryogenic conditions. The application of serial crystallography for studying protein dynamics is mentioned.

Key words

Membrane protein GPCR X-ray Serial Dynamics Conformation Crystallography Lipidic cubic phase LCP Injector Rhodopsin 

Notes

Acknowledgments

We thank Thomas Gruhl for helping in developing the present protocol for rhodopsin serial crystallography and providing some pictures. We thank Edwards Stuttfeld for the continuous support and advices to work with the SONICC imager, and Timm Maier for the access to the SONICC imager. We acknowledge the Paul Scherrer Institut, Villigen, Switzerland, for provision of synchrotron radiation beam time at beamline PXI of the SLS and the continuous support from Andrea Prota and Takashi Tomizaki. This work was supported by the NCCR Molecular Systems Engineering 2015–2017 and Swiss National Science Foundation grant 173335 (to Gebhard Schertler). We acknowledge Filip Pamula and Maximilian Wranik for critical reading of the manuscript.

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Authors and Affiliations

  1. 1.Division of Biology and Chemistry, Laboratory of Biomolecular ResearchPaul Scherrer InstituteVilligen PSISwitzerland

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