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Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography

Nature Protocols volume 9, pages 21232134 (2014) | Download Citation

Abstract

We have recently established a procedure for serial femtosecond crystallography (SFX) in lipidic cubic phase (LCP) for protein structure determination at X-ray free-electron lasers (XFELs). LCP-SFX uses the gel-like LCP as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room-temperature high-resolution data with minimal effects of radiation damage from sub-10-μm crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared with SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2–3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3–5 d, including the time required for the crystals to grow.

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Acknowledgements

This work was supported by the US National Institutes of Health grant nos. P50 GM073197 and U54 GM094618. We thank K. Kadyshevskaya for assistance with figure preparation, L. Johansson for comments and A. Walker for assistance with manuscript preparation.

Author information

Affiliations

  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.

    • Wei Liu
    • , Andrii Ishchenko
    •  & Vadim Cherezov
  2. Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening and Marine Drug Research Institute, Huaihai Institute of Technology, Lianyungang, China.

    • Wei Liu

Authors

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Contributions

W.L. and A.I. worked out the protocols and wrote the initial draft, and V.C. developed the concept and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Vadim Cherezov.

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DOI

https://doi.org/10.1038/nprot.2014.141

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