UNIT 17.13 Cryo-Electron Tomography for Structural Characterization of Macromolecular Complexes
Published Online: 1 AUG 2011
Copyright © 2011 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Cope, J., Heumann, J. and Hoenger, A. 2011. Cryo-Electron Tomography for Structural Characterization of Macromolecular Complexes. Current Protocols in Protein Science. 65:17.13:17.13.1–17.13.31.
- Published Online: 1 AUG 2011
- Published Print: AUG 2011
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. Curr. Protoc. Protein Sci. 65:17.13.1-17.13.31. © 2011 by John Wiley & Sons, Inc.
- cryo-electron microscopy (cryo-EM);
- cryo-electron tomography (cryo-ET);
- vitrification of macromolecules;
- tilt-series acquisition;
- sub-volume averaging