ジャーナル: Nat Commun / 年: 2020 タイトル: Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM. 著者: Ricardo M Sanchez / Yingyi Zhang / Wenbo Chen / Lea Dietrich / Mikhail Kudryashev / 要旨: 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 ...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.
EMPIAR-10452 (タイトル: Cryo Electron Tomograms of Membrane Fractions of Rabbit Skeletal Muscle for Structural Determination of RyR1 in SR Vesicles at subnanometer resolution Data size: 435.8 Data #1: 48 tilt series (frames aligned my motioncorr2) in .st stacks (mrc format for imod) including .tlt, .xf and .defocus files to generate reconstructions [tilt series])