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High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity

Nature Protocols volume 17, pages 421–444 (2022)Cite this article

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Abstract

Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.

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Fig. 1: emClarity processing workflow.
Fig. 2: Template matching.
Fig. 3: 3D classification and sampling function.
Fig. 4: Subtomogram averages and conical FSC plots of HIV-1 Gag T8I assemblies (five tilt series).
Fig. 5: STA of WT Gag (five tilt series, EMPIAR-10164).
Fig. 6: Subtomogram classification and averaging of ribosome (12 tilt series, EMPIAR-10304).
Fig. 7: STA of apoferritin (six tilt series).

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Data availability

The Gag dataset (five tilt series) and apoferritin dataset (six tilt series) have been deposited in the EMPIAR database under accession codes EMPIAR-10643 and EMPIAR-10787, respectively. The resulting final reconstructions have been deposited in EMDB under the following accession codes: Gag-T8I, EMD-13390; Gag-WT, EMD-13354; apoferritin, EMD-13271; and ribosome, EMD-13270.

Code availability

The emClarity software is freely available at https://github.com/bHimes/emClarity/wiki. The tutorial documentation is available at https://github.com/ffyr2w/emClarity-tutorial.

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Acknowledgements

We are grateful to Y. Zhu for discussion and critical reading of the manuscript. We acknowledge Diamond for access and support of the CryoEM facilities at the UK national Electron Bio-Imaging Centre (eBIC, proposal CM26464), funded by the Wellcome Trust, Medical Research Council (MRC) and Biotechnology and Biological Sciences Research Council (BBSRC). The computational aspects of this research were supported by the Wellcome Trust Core Award grant number 203141/Z/16/Z and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). This work was supported by the National Institutes of Health grants AI150481, the UK Wellcome Trust Investigator Award 206422/Z/17/Z, the UK Biotechnology and Biological Sciences Research Council grant BB/S003339/1, and the European Research Council Advanced Grant (ERC AdG) grant 101021133.

Author information

Author notes

  1. These authors contributed equally: Tao Ni, Thomas Frosio, Luiza Mendonça.

Authors and Affiliations

  1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Tao Ni, Thomas Frosio, Luiza Mendonça & Peijun Zhang

  2. Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK

    Thomas Frosio, Yuewen Sheng, Daniel Clare & Peijun Zhang

  3. Howard Hughes Medical Institute, RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Benjamin A. Himes

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Contributions

P.Z. conceived the research and designed the experiments. Y.S. prepared the apoferritin on graphene grids, and D.C. collected data. T.N., L.M. and Y.S. performed tomography reconstruction and STA and classification. T.F. wrote the emClarity tutorial. B.A.H. and T.F. updated code/binaries with new features in later versions of emClarity. T.N. and P.Z. wrote the manuscript with support from all the authors.

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Correspondence to Peijun Zhang.

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The authors declare no competing interests.

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Nature Protocols thanks Peter J. Peters and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Liu, C. et al. Structure 28, 1218–1224.e1214 (2020): https://doi.org/10.1016/j.str.2020.10.001

Watanabe, R. et al. Cell 182, 1508–1518.e1516 (2020): https://doi.org/10.1016/j.cell.2020.08.004

Sutton, G. et al. Nat. Commun. 11, 4445 (2020): https://doi.org/10.1038/s41467-020-18243-9

Tan, T. Y. et al. Nat. Commun. 11, 895 (2020): https://doi.org/10.1038/s41467-020-14647-9

Unchwaniwala, N. et al. Proc. Natl Acad. Sci. USA 117, 18680–18691 (2020): https://doi.org/10.1073/pnas.2006165117

Gibson, K. H. et al. eLife 9, e53672 (2020): https://doi.org/10.7554/eLife.53672

Cassidy, C. K. et al. Commun. Biol. 3, 24 (2020): https://doi.org/10.1038/s42003-019-0748-0

Key data used in this protocol

Eisenstein, F. et al. J. Struct. Biol. 208, 107–114 (2019): https://doi.org/10.1016/j.jsb.2019.08.006

Schur, F. K. et al. Science 353 506–508 (2016): https://doi.org/10.1126/science.aaf9620

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Ni, T., Frosio, T., Mendonça, L. et al. High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity. Nat Protoc 17, 421–444 (2022). https://doi.org/10.1038/s41596-021-00648-5

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