Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM
- 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.
Author: | Ricardo Miguel Sánchez LoayzaORCiD, Yingyi ZhangORCiDGND, Wenbo ChenGND, Lea DietrichORCiDGND, Mikhail KudryashevORCiDGND |
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URN: | urn:nbn:de:hebis:30:3-798977 |
DOI: | https://doi.org/10.1038/s41467-020-17466-0 |
ISSN: | 2041-1723 |
Parent Title (English): | Nature Communications |
Publisher: | Nature Publishing Group UK |
Place of publication: | London |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2020/07/24 |
Date of first Publication: | 2020/07/24 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2024/04/10 |
Volume: | 11 |
Issue: | 3709 |
Article Number: | 3709 |
Page Number: | 7 |
Institutes: | Angeschlossene und kooperierende Institutionen / MPI für Biophysik |
Fachübergreifende Einrichtungen / Buchmann Institut für Molekulare Lebenswissenschaften (BMLS) | |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - Namensnennung 4.0 |