In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2023 Feb 26

PMID 36842118

Authors

Longsheng Lai

Yee-Wai Cheung

Matthew Martinez

Kathryn Kixmoeller

Leon Palao 3rd

Stefan Steimle

Meng-Chiao Ho

Ben E Black

Erh-Min Lai

Yi-Wei Chang

Affiliations

Soon will be listed here.

Abstract

Bacterial surface nanomachines are often refractory to structural determination in their intact form due to their extensive association with the cell envelope preventing them from being properly purified for traditional structural biology methods. Cryo-electron tomography (cryo-ET) is an emerging branch of cryo-electron microscopy that can visualize supramolecular complexes directly inside frozen-hydrated cells in 3D at nanometer resolution, therefore posing a unique capability to study the intact structures of bacterial surface nanomachines in situ and reveal their molecular association with other cellular components. Furthermore, the resolution of cryo-ET is continually improving alongside methodological advancement. Here, using the type IV pilus machine in Myxococcus xanthus as an example, we describe a step-by-step workflow for in situ structure determination including sample preparation and screening, microscope and camera tuning, tilt series acquisition, data processing and tomogram reconstruction, subtomogram averaging, and structural analysis.

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