Sep 30, 2016

Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION

Nature Protocols
Tanmay A M Bharat, Sjors H W Scheres

Abstract

Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis. We describe herein a protocol for subtomogram averaging from cryo-ET data using the RELION software (http://www2.mrc-lmb.cam.ac.uk/relion). RELION was originally developed for cryo-EM single-particle analysis, and the subtomogram averaging approach presented in this protocol has been implemented in the existing workflow for single-particle analysis so that users may conveniently tap into existing capabilities of the RELION software. We describe how to calculate 3D models for the contrast transfer function (CTF) that describe the transfer of information in the imaging process, and we illustrate the results of classification and subtomogram averaging refinement for cryo-ET data of purified ...Continue Reading

  • References38
  • Citations32

References

  • References38
  • Citations32

Citations

Mentioned in this Paper

Electron Microscope Tomography
Computer Software
Macromolecular Compounds
Classification
Virus
Saccharomyces cerevisiae allergenic extract
Complex (molecular entity)
Determination Aspects
Dane Particle
X-Ray Computed Tomography