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In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography

  • Gregor L. Weiss
  • João M. Medeiros
  • Martin Pilhofer
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

The unique property of electron cryotomography (ECT) is its capability to resolve the structure of macromolecular machines in their cellular context. The integration of ECT data with high-resolution structures of purified subcomplexes and live-cell fluorescence light microscopy can generate pseudo-atomic models that lead to a mechanistic understanding across size and time scales. Recent advances in electron detection, sample thinning, data acquisition, and data processing have significantly enhanced the applicability and performance of ECT. Here we describe a detailed workflow for an ECT experiment, including cell culture, vitrification, data acquisition, data reconstruction, tomogram analysis, and subtomogram averaging. This protocol provides an entry point to the technique for students and researchers and indicates the many possible variations arising from specific target properties and the available instrumentation.

Key words

Tilt series Plunge freezing Electron cryomicroscopy Reconstruction Cryogen Segmentation 

Notes

Acknowledgments

We thank D. Böck, R. Kooger, and P. Szwedziak for comments on the manuscript. G. L. Weiss was supported by a Boehringer Ingelheim Fonds PhD Fellowship. The Pilhofer Lab is supported by grants from ETH Zürich, the European Research Council, the Swiss National Science Foundation, and the Helmut Horten Foundation.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Gregor L. Weiss
    • 1
  • João M. Medeiros
    • 1
  • Martin Pilhofer
    • 1
  1. 1.Department of Biology, ETH ZürichInstitute of Molecular Biology and BiophysicsZürichSwitzerland

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