title: Specimen Preparation for High-Resolution Cryo-EM.
abstract: Imaging a material with electrons at near-atomic resolution requires a thin specimen that is stable in the vacuum of the transmission electron microscope. For biological samples, this comprises a thin layer of frozen aqueous solution containing the biomolecular complex of interest. The process of preparing a high-quality specimen is often the limiting step in the determination of structures by single-particle electron cryomicroscopy (cryo-EM). Here, we describe a systematic approach for going from a purified biomolecular complex in aqueous solution to high-resolution electron micrographs that are suitable for 3D structure determination. This includes a series of protocols for the preparation of vitrified specimens on various supports, including all-gold and graphene. We also describe techniques for troubleshooting when a preparation fails to yield suitable specimens, and common mistakes to avoid during each part of the process. Finally, we include recommendations for obtaining the highest quality micrographs from prepared specimens with current microscope, detector, and support technology.
authors: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. Electronic address: passmore@mrc-lmb.cam.ac.uk.
authors: L A
authors: LA
authors: Passmore
authors: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. Electronic address: crusso@mrc-lmb.cam.ac.uk.
authors: C J
authors: CJ
authors: Russo
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D006046:Gold / Q000737:chemistry*; D006108:Graphite / Q000737:chemistry*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D011506:Proteins / Q000648:ultrastructure; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D058989:Vitrification
chemical_list: D011506:Proteins; D006046:Gold; D006108:Graphite
keywords: Cryo-EM; Electron cryo-microscopy; Electron tomography; Protein structure; Single-particle reconstruction; Substrates