Abstract
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.