Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs.

Autor: Obr M; Institute of Science and Technology Austria (ISTA), Klosterneuburg, Austria., Hagen WJH; European Molecular Biology Laboratory (EMBL), Heidelberg, Germany., Dick RA; Department of Molecular Biology and Genetics, Cornell University, Ithaca, USA., Yu L; Materials and Structural Analysis Division, Thermo Fisher Scientific, Eindhoven, Netherlands., Kotecha A; Materials and Structural Analysis Division, Thermo Fisher Scientific, Eindhoven, Netherlands., Schur FKM; Institute of Science and Technology Austria (ISTA), Klosterneuburg, Austria. Electronic address: florian.schur@ist.ac.at.
Jazyk: angličtina
Zdroj: Journal of structural biology [J Struct Biol] 2022 Jun; Vol. 214 (2), pp. 107852. Date of Electronic Publication: 2022 Mar 26.
DOI: 10.1016/j.jsb.2022.107852
Abstrakt: The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.
(Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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