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

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.

SUBMITTER: Sanchez RM 

PROVIDER: S-EPMC7381653 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature