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标题：Cryo-EM grid optimization for membrane proteins
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作者：Domen Kampjut ; Julia Steiner ; Leonid A. Sazanov 等
期刊名称：iScience
印刷版ISSN：2589-0042
出版年度：2021
卷号：24
期号：3
页码：1-17
DOI：10.1016/j.isci.2021.102139
语种：English
出版社：Elsevier
摘要：SummaryCryo-EM grid preparation is an important bottleneck in protein structure determination, especially for membrane proteins, typically requiring screening of a large number of conditions. We systematically investigated the effects of buffer components, blotting conditions and grid types on the outcome of grid preparation of five different membrane protein samples. Aggregation was the most common type of problem which was addressed by changing detergents, salt concentration or reconstitution of proteins into nanodiscs or amphipols. We show that the optimal concentration of detergent is between 0.05 and 0.4% and that the presence of a low concentration of detergent with a high critical micellar concentration protects the proteins from denaturation at the air-water interface. Furthermore, we discuss the strategies for achieving an adequate ice thickness, particle coverage and orientation distribution on free ice and on support films. Our findings provide a clear roadmap for comprehensive screening of conditions for cryo-EM grid preparation of membrane proteins.Graphical abstractDisplay OmittedHighlights•Cryo-EM grid optimization is a limiting factor in protein structure determination•Membrane proteins require additional optimization of parameters such as surfactants•Aggregation and intactness of proteins depend on detergent and salt concentrations•Support films can help preserve protein intactness and improve particle coverageBiochemistry; Structural Biology