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Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM

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Item Type:Article
Title:Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM
Creators Name:Sanchez, R.M. and Zhang, Y. and Chen, W. and Dietrich, L. and Kudryashev, M.
Abstract:Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.
Keywords:Calcium Channels, Cryoelectron Microscopy, Electron Microscope Tomography, Computer-Assisted Image Processing, Macromolecular Substances, Ryanodine Receptor Calcium Release Channel, Signal-To-Noise Ratio, Single Molecule Imaging, Workflow
Source:Nature Communications
Publisher:Nature Publishing Group
Page Range:3709
Date:24 July 2020
Official Publication:https://doi.org/10.1038/s41467-020-17466-0
PubMed:View item in PubMed

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