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

Nature Protocols volume 11, pages 2054–2065 (2016)Cite this article

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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 hepatitis B capsid particles and Saccharomyces cerevisiae 80S ribosomes. Using the steps described in this protocol, along with the troubleshooting and optimization guidelines, high-resolution maps can be obtained in which secondary structure elements are resolved subtomogram.

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Figure 1: Workflow of the image processing protocol.
Figure 2: CTF estimation for the 3D CTF model.
Figure 3: The RELION-1.4 graphical user interface.
Figure 4: 2D Classification and initial model generation.
Figure 5: 3D auto-refinement and classification using the regularized-likelihood algorithm in RELION.
Figure 6: Subtomogram analysis of particles at different Z heights.

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Acknowledgements

We thank X.C. Bai, I.S. Fernandez and K. Vinothhumar for help with sample preparation; J. Grimmett and T. Darling for assistance with high-performance computing; S. Chen and C. Savva for assistance with electron microscopy; and J. Löwe for helpful discussions. This work was supported by funds from EMBO (ALTF 3-2013 and aALTF 778-2015 to T.A.M.B.) and the UK Medical Research Council (MC_UP_A025_1013 to S.H.W.S.).

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Authors and Affiliations

  1. Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK

    Tanmay A M Bharat & Sjors H W Scheres

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  1. Tanmay A M Bharat
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  2. Sjors H W Scheres
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Contributions

T.A.M.B. performed tomographic data acquisition and data processing, and developed the Python script for pre-processing of the subtomograms. S.H.W.S. developed the subtomogram procedures inside RELION. Both authors contributed to writing the manuscript.

Corresponding authors

Correspondence to Tanmay A M Bharat or Sjors H W Scheres.

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The authors declare no competing financial interests.

Additional information

The data set of seven tomograms of 80S ribosomes from S. cerevisiae has been deposited at the EMPIAR database (EMPIAR-10045), and the final map from 3D auto-refinement of these data has been submitted to the EMDB (EMD-3228).

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Bharat, T., Scheres, S. Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION. Nat Protoc 11, 2054–2065 (2016). https://doi.org/10.1038/nprot.2016.124

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