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

Ricardo M. Sanchez, Yingyi Zhang, Wenbo Chen, Lea Dietrich and Mikhail Kudryashev ()
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Ricardo M. Sanchez: Max Planck Institute for Biophysics
Yingyi Zhang: Max Planck Institute for Biophysics
Wenbo Chen: Max Planck Institute for Biophysics
Lea Dietrich: Max Planck Institute for Biophysics
Mikhail Kudryashev: Max Planck Institute for Biophysics

Nature Communications, 2020, vol. 11, issue 1, 1-7

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-17466-0

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