title: Cryo-electron tomography for structural characterization of macromolecular complexes.
abstract: Cryo-electron tomography (cryo-ET) is an emerging 3-D reconstruction technology that combines the principles of tomographic 3-D reconstruction with the unmatched structural preservation of biological matter embedded in vitreous ice. Cryo-ET is particularly suited to investigating cell-biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging-based 3-D reconstruction procedures. This unit aims to make cryo-ET accessible to newcomers and discusses the specialized equipment required, as well as relevant advantages and hurdles associated with sample preparation by vitrification and cryo-ET. Protocols describe specimen preparation, data recording and 3-D data reconstruction for cryo-ET, with a special focus on macromolecular complexes. A step-by-step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt-series acquisition for cryo-ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo-tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal-to-noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Cope
authors.forename: John
authors.initials: J
authors.lastname: Heumann
authors.forename: Andreas
authors.initials: A
authors.lastname: Hoenger
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D055032:Electron Microscope Tomography / Q000295:instrumentation* / Q000379:methods*; D046911:Macromolecular Substances / Q000737:chemistry*
chemical_list: D046911:Macromolecular Substances