TY  - INPR
A1  - Sánchez Loayza, Ricardo Miguel
A1  - Zhang, Yingyi
A1  - Chen, Wenbo
A1  - Dietrich, Lea
A1  - Kudryashev, Mikhail
T1  - Subnanometer-resolution structure determination in situ by a hybrid subtomogram averaging single particle cryoEM workflow
T2  - bioRxiv
N2  - Cryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.
Y1  - 2020
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/72735
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-727357
UR  - https://www.biorxiv.org/content/10.1101/2020.04.24.057299v1
IS  - 2020.04.24.057299 Version 1
PB  - bioRxiv
ER  -