Apr 25, 2020

Subnanometer-resolution structure determination in situ by a hybrid subtomogram averaging - single particle cryoEM - workflow

BioRxiv : the Preprint Server for Biology
R. SanchezMisha Kudryashev

Abstract

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 Angstrom, 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 Angstrom and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Angstrom. We demonstrate that hStA can improve the ...Continue Reading

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Mentioned in this Paper

Establishment and Maintenance of Localization
Place Cells
Cytoplasmic Vesicles
Pathogenic Organism
Actins
Virion
Spatial Distribution
Gene Expression
Site
Local

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