Howard Hughes Medical Institute, University of California Los Angeles; Department of Biological Chemistry, University of California Los Angeles.
Howard Hughes Medical Institute, University of California Los Angeles; Department of Biological Chemistry, University of California Los Angeles; Department of Physiology, University of California Los Angeles;
J Vis Exp. 2021 Mar 15(169). doi: 10.3791/62313.
A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.
描述了一种用于将小分子样品制备用于微晶体电子衍射（MicroED）实验的详细方案。MicroED 已经被开发出来，以便使用标准的电子冷冻显微镜（cryo-EM）设备来解决蛋白质和小分子的结构问题。通过这种方式，小分子、肽、可溶性蛋白质和膜蛋白最近已经被确定具有高分辨率。这里提供了使用药物卡马西平作为示例来制备小分子药物的网格的方案。还介绍了筛选和收集数据的方案。整个过程中的其他步骤，如数据集成、结构确定和精修，在其他地方介绍。估计准备小分子网格所需的时间少于 30 分钟。
