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• W3128680360 abstract "Cryo-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." @default.
• W3128680360 created "2021-02-15" @default.
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• W3128680360 date "2021-03-01" @default.
• W3128680360 modified "2023-10-17" @default.
• W3128680360 title "Cryo-EM grid optimization for membrane proteins" @default.
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• W3128680360 doi "https://doi.org/10.1016/j.isci.2021.102139" @default.
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