1. Structural Biology and Molecular Biophysics
Download icon

Fully automated, sequential focused ion beam milling for cryo-electron tomography

  1. Tobias Zachs
  2. Andreas Schertel
  3. João Medeiros
  4. Gregor L Weiss
  5. Jannik Hugener
  6. Joao Matos
  7. Martin Pilhofer  Is a corresponding author
  1. ETH Zürich, Switzerland
  2. Carl Zeiss Microscopy GmbH, Germany
Tools and Resources
  • Cited 2
  • Views 1,956
  • Annotations
Cite this article as: eLife 2020;9:e52286 doi: 10.7554/eLife.52286

Abstract

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

Article and author information

Author details

  1. Tobias Zachs

    Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  2. Andreas Schertel

    Zeiss Customer Center Europe, Carl Zeiss Microscopy GmbH, Oberkochen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. João Medeiros

    Department of Biology, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9075-548X
  4. Gregor L Weiss

    Department of Biology, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  5. Jannik Hugener

    Department of Biology, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  6. Joao Matos

    Institute of Biochemistry, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3754-3709
  7. Martin Pilhofer

    Department of Biology, ETH Zürich, Zürich, Switzerland
    For correspondence
    pilhofer@biol.ethz.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3649-3340

Funding

Swiss National Science Foundation (#31003A_179255)

  • Martin Pilhofer

European Research Council (#679209)

  • Martin Pilhofer

Nomis Foundation (nd)

  • Martin Pilhofer

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom

Publication history

  1. Received: September 28, 2019
  2. Accepted: March 7, 2020
  3. Accepted Manuscript published: March 9, 2020 (version 1)
  4. Version of Record published: March 19, 2020 (version 2)

Copyright

© 2020, Zachs et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,956
    Page views
  • 332
    Downloads
  • 2
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Structural Biology and Molecular Biophysics
    Tone Bengtsen et al.
    Research Article

    Nanodiscs are membrane mimetics that consist of a protein belt surrounding a lipid bilayer, and are broadly used for characterization of membrane proteins. Here, we investigate the structure, dynamics and biophysical properties of two small nanodiscs, MSP1D1ΔH5 and ΔH4H5. We combine our SAXS and SANS experiments with molecular dynamics simulations and previously obtained NMR and EPR data to derive and validate a conformational ensemble that represents the structure and dynamics of the nanodisc. We find that it displays conformational heterogeneity with various elliptical shapes, and with substantial differences in lipid ordering in the centre and rim of the discs. Together, our results reconcile previous apparently conflicting observations about the shape of nanodiscs, and paves the way for future integrative studies of larger complex systems such as membrane proteins embedded in nanodiscs.

    1. Structural Biology and Molecular Biophysics
    Sigrid Noreng et al.
    Research Advance

    The molecular bases of heteromeric assembly and link between Na+ self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated that ENaC subunits – α, β, and γ – assemble in a counterclockwise configuration when viewed from outside the cell with the protease-sensitive GRIP domains in the periphery (Noreng et al., 2018). Here we describe the structure of ENaC resolved by cryo-electron microscopy at 3 Å. We find that a combination of precise domain arrangement and complementary hydrogen bonding network defines the subunit arrangement. Furthermore, we determined that the α subunit has a primary functional module consisting of the finger and GRIP domains. The module is bifurcated by the α2 helix dividing two distinct regulatory sites: Na+ and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na+ site via the α2 helix highlighting the critical role of the α2 helix in regulating ENaC function.