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Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms

Nature Methods volume 17, pages 209–216 (2020)Cite this article

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An Author Correction to this article was published on 27 January 2020

An Author Correction to this article was published on 22 January 2020

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Abstract

With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ.

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Fig. 1: Tracing density at a synapse from rodent cerebrocortical synaptosomal preparation.
Fig. 2: Method validations.
Fig. 3: Processing of ER membrane-associated complexes from the microsomal dataset.
Fig. 4: Complexes resolved in situ from the intact P19 cells dataset.
Fig. 5: Spatial distribution of microsomal particles.

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Data availability

The following electron tomography densities have been deposited in the EMDataBank. Subtomogram averages obtained from the microsomal dataset, cytosolic particles: ribosome bound to the fully assembled (EMD-0074) and partial translocon complex (EMD-0084), the large ribosomal subunit (EMD-0075), and obtained from microsomal dataset lumenal particles: the ribosome-translocon complex (EMD-0085), the ribosome-free fully assembled translocon (EMD-0086) and the nontranslocon-associated OST complex (EMD-0087). Microsomal tomograms EMD-10449, EMD-10450 and EMD-10451. Subtomogram averages obtained from the in situ P19 cells dataset: membrane-associated ribosome (EMD-10432), ribosome-associated translocon (EMD-10433), ribosome-free translocon (EMD-10434), two putative PLC complexes (EMD-10435, EMD-10436), putative IP3 receptor (EMD-10437). In situ P19 cells tomogram shown in Fig. 4EMD-10439).

Source data for Fig. 2 is available with the paper. Data for Supplementary Fig. 4 are available from the corresponding authors upon request.

Code availability

The complete software, together with all dependencies, is installed as PySeg capsule on Code Ocean67 (https://doi.org/10.24433/CO.0526052.v2). The latest version of the software is available upon demand and on GitHub (https://github.com/anmartinezs/pyseg_system.git).

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Acknowledgements

We thank F. Beck for useful discussions and G. J. Greif for critical reading of the manuscript. A.M.-S. was the recipient of a postdoctoral fellowship from the Séneca Foundation. This work was supported by the European Commission (grant no. FP7 GA ERC-2012-SyG_318987–ToPAG) and by Max Planck Society.

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Authors and Affiliations

  1. Max Planck Institute of Biochemistry, Martinsried, Germany

    Antonio Martinez-Sanchez, Zdravko Kochovski, Ulrike Laugks, Johannes Meyer zum Alten Borgloh, Saikat Chakraborty, Stefan Pfeffer, Wolfgang Baumeister & Vladan Lučić

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Contributions

A.M.-S. and V.L. conceived and designed the research. A.M.-S. designed and implemented the software. Z.K., U.L. and S.C. acquired original tomograms. S.P. provided expertise related to the previously recorded tomograms. A.M.-S., J.M.z.A.B. and V.L. analyzed the data. W.B. provided resources and acquired funding. V.L. supervised research. A.M.-S. and V.L. wrote the manuscript. All authors edited the manuscript.

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Correspondence to Antonio Martinez-Sanchez or Vladan Lučić.

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Peer review information Allison Doerr was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary Figs. 1–11

Reporting Summary

Supplementary Video 1

Detection of microsome-attached complexes and localization of lumenal particles Density minima are shown as small spheres (red, cytosolic; blue, membrane; green, lumenal), arcs as gray lines and green arrows denote membrane normal vectors. N = 55 tomograms.

Supplementary Video 2

Localization of microsome-attached complexes Ribosomes derived from the cytosolic particles are shown in red, ribosome-free fully assembled translocon complexes in blue and the nontranslocon-associated OST complexes in yellow. N = 55 tomograms.

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Martinez-Sanchez, A., Kochovski, Z., Laugks, U. et al. Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms. Nat Methods 17, 209–216 (2020). https://doi.org/10.1038/s41592-019-0675-5

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