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Harrison, Peter J.; Vecerkova, Tereza; Clare, Daniel K.; Quigley, Andrew
Journal of structural biology, June 2023, 2023-06-00, 20230601, Volume: 215, Issue: 2Journal Article
Display omitted •Most sub-100 kDa membrane proteins solved by cryo-EM are encapsulated in detergent.•DDM is the most popular detergent for small membrane proteins.•Carbon on gold grids are the most popular for sub- 100 kDa membrane proteins.•Many different approaches are taken for data processing. Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structurally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity as well as conformational and compositional instability. Here we have reviewed the sample preparation approaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM (those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.
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