Optimization of Sample Preparation for the Observation of Macromolecular Complexes by Electron (cryo-)Microscopy.
Fiche publication
Date publication
janvier 2021
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr SCHULTZ Patrick
Tous les auteurs :
Frechard A, Sharov G, Werderer M, Schultz P
Lien Pubmed
Résumé
Electron microscopy is a powerful tool for studying the homogeneity and structure of biomolecular complexes. The small wavelength of electron and the availability of electron optics enable the direct visualization of macromolecular assemblies in a large range of sizes between 5 and 100 nm. This informs us about the degree of multimerization or aggregation and provides precise information about their general shape and dimensions. When combined with sophisticated image analysis protocols, three-dimensional (3D) information can be gained from 2D projections of the sample, leading to a structural description. When intermediate steps of a reaction can be imaged, insights into the mode of action of macromolecules can be gained, and structure-function relations can be established. However, the way the sample is prepared for its observation within the vacuum of an electron microscope determines the information that can be retrieved from the experiment. We will review two commonly used specimen preparation protocols for subsequent single-particle electron microscopy observation, namely negative staining and vitrification.
Mots clés
Cryo-EM, EM sample preparation, Negative stain electron microscopy, Single particle electron microscopy
Référence
Methods Mol Biol. 2021 ;2247:243-256