title: Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography.
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.
authors.affiliation: 1] Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA. [2] Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening and Marine Drug Research Institute, Huaihai Institute of Technology, Lianyungang, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Andrii
authors.initials: A
authors.lastname: Ishchenko
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Vadim
authors.identifier: 0000000252653914
authors.initials: V
authors.lastname: Cherezov
mesh_terms: D003461:Crystallography / Q000379:methods*; D007834:Lasers; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D013997:Time Factors
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins