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TitleSubnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM.
Journal, issue, pagesNat Commun, Vol. 11, Issue 1, Page 3709, Year 2020
Publish dateJul 24, 2020
AuthorsRicardo M Sanchez / Yingyi Zhang / Wenbo Chen / Lea Dietrich / Mikhail Kudryashev /
PubMed AbstractCryo-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.
External linksPubMed:32709843 / Publisher's page
MethodsEM (subtomogram averaging)
Resolution5.24 - 9.1 A
Structure data

EMDB-10834:
Tobacco mosaic virus (TMV) structure determined by a hybrid STA-SPA workflow

EMDB-10840:
Structure of RyR1 in apo/closed state in native membrane

Source
  • Tobacco mosaic virus
  • Oryctolagus cuniculus (rabbit)

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