title: The collection of MicroED data for macromolecular crystallography.
abstract: The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Shi
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Brent L
authors.initials: BL
authors.lastname: Nannenga
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: M Jason
authors.initials: MJ
authors.lastname: de la Cruz
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Jinyang
authors.initials: J
authors.lastname: Liu
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Sawtelle
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Calero
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Francis E
authors.initials: FE
authors.lastname: Reyes
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Johan
authors.identifier: http://orcid.org/0000-0002-8936-0912
authors.initials: J
authors.lastname: Hattne
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Tamir
authors.initials: T
authors.lastname: Gonen
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D003460:Crystallization; D003461:Crystallography / Q000379:methods*; D003625:Data Collection / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods; D004867:Equipment Design; D007091:Image Processing, Computer-Assisted / Q000379:methods; D046911:Macromolecular Substances / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012984:Software
chemical_list: D046911:Macromolecular Substances; D011506:Proteins