Jul 2, 2017

In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography

Methods in Molecular Biology
Gregor L WeissMartin Pilhofer


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.

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Mentioned in this Paper

NDUFA5 protein, human
Reconstructive Surgical Procedures
Pseudo brand of pseudoephedrine
Cell Culture Techniques
Research Personnel
Bacterial Secretion Systems
Process of Secretion
Optical Image Reconstruction