title: Electron Microscopy and Image Processing: Essential Tools for Structural Analysis of Macromolecules.
abstract: Macromolecular electron microscopy typically depicts the structures of macromolecular complexes ranging from ∼200 kDa to hundreds of MDa. The amount of specimen required, a few micrograms, is typically 100 to 1000 times less than needed for X-ray crystallography or nuclear magnetic resonance spectroscopy. Micrographs of frozen-hydrated (cryogenic) specimens portray native structures, but the original images are noisy. Computational averaging reduces noise, and three-dimensional reconstructions are calculated by combining different views of free-standing particles ("single-particle analysis"). Electron crystallography is used to characterize two-dimensional arrays of membrane proteins and very small three-dimensional crystals. Under favorable circumstances, near-atomic resolutions are achieved. For structures at somewhat lower resolution, pseudo-atomic models are obtained by fitting high-resolution components into the density. Time-resolved experiments describe dynamic processes. Electron tomography allows reconstruction of pleiomorphic complexes and subcellular structures and modeling of macromolecules in their cellular context. Significant information is also obtained from metal-coated and dehydrated specimens.
authors.affiliation: Departments of Biology and Biochemistry, University of Utah, Salt Lake City, Utah.
authors.forename: David M
authors.initials: DM
authors.lastname: Belnap
mesh_terms: D000818:Animals; D006225:Hand / Q000000981:diagnostic imaging; D006430:Hemiptera; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional; D046911:Macromolecular Substances / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D011859:Radiography
chemical_list: D046911:Macromolecular Substances
keywords: cryo-electron microscopy; cryogenic electron microscopy; direct electron detector; electron cryo-microscopy; electron crystallography; electron tomography; frozen-hydrated specimen; immunolabeling; macromolecular complex; metal shadowing; negative stain; single-particle analysis; three-dimensional electron microscopy; three-dimensional image reconstruction; tomography; transmission electron microscopy; two-dimensional crystal; vitreous ice