Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED.
Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED.
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DOI：
10.1007/978-1-0716-1406-8_16
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发表时间：
2021
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微晶电子衍射（MicroED）是最新的冷冻电子显微镜（cryo-EM）方法，已经确定了70多种蛋白质，肽和几种小有机分子的结构。在MicroED中，溶液中的微米或纳米晶样品沉积在电子显微镜网格上，并在低温电子显微镜中进行检查，理想情况下在低温条件下进行。收集连续旋转衍射数据，然后使用常规X射线晶体学程序进行处理。这里概述的协议详细介绍了如何获得和识别纳米晶体，如何设置显微镜进行筛选和MicroED数据收集，以及如何收集和处理数据以完成高分辨率结构。对于在cryo-EM中具有高分辨率衍射的性能良好的晶体，整个过程可以在不到一个小时的时间内完成。
Microcrystal Electron Diffraction (MicroED) is the newest cryo-electron microscopy (cryo-EM) method, with over 70 protein, peptide, and several small organic molecule structures already determined. In MicroED, micro- or nanocrystalline samples in solution are deposited on electron microscopy grids and examined in a cryo-electron microscope, ideally under cryogenic conditions. Continuous rotation diffraction data are collected and then processed using conventional X-ray crystallography programs. The protocol outlined here details how to obtain and identify the nanocrystals, how to set up the microscope for screening and for MicroED data collection, and how to collect and process data to complete high-resolution structures. For well-behaving crystals with high-resolution diffraction in cryo-EM, the entire process can be achieved in less than an hour.