Membrane Protein Cryo-EM: Cryo-Grid Optimization and Data Collection with Protein in Detergent
Membrane Protein Cryo-EM: Cryo-Grid Optimization and Data Collection with Protein in Detergent
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DOI：
10.1007/978-1-0716-0373-4_16
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发表时间：
2020-01-01
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影响因子：
--
通讯作者：
Taylor, Nicholas M. I.
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文献类型：
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作者：
Bloch, Magnus;Santiveri, Nica;Taylor, Nicholas M. I.
低温电子显微镜(Cryo-EM)是在近自然条件下研究大分子结构的有力工具。特别是在膜蛋白的背景下，这项技术使研究人员能够获得以前无法获得的详细程度的结构信息。样品制备仍然是大多数低温EM研究项目的瓶颈，膜蛋白是特别具有挑战性的研究对象，因为它们普遍需要洗涤剂或其他增溶剂。在这里，我们描述了负染和低温EM网格的制备以及洗涤剂中膜蛋白的下游数据收集，洗涤剂是迄今为止最常见的增溶剂。该方案概述了一种在生物学相关条件下筛选和确定感兴趣的膜蛋白结构的快速而直接的程序。
Cryo-electron microscopy (cryo-EM) is a powerful tool for investigating the structure of macromolecules under near-native conditions. Especially in the context of membrane proteins, this technique has allowed researchers to obtain structural information at a previously unattainable level of detail. Specimen preparation remains the bottleneck of most cryo-EM research projects, with membrane proteins representing particularly challenging targets of investigation due to their universal requirement for detergents or other solubilizing agents. Here we describe preparation of negative staining and cryo-EM grids and downstream data collection of membrane proteins in detergent, by far the most common solubilization agent. This protocol outlines a quick and straightforward procedure for screening and determining the structure of a membrane protein of interest under biologically relevant conditions.