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

View ORCID ProfileMartin Obr, View ORCID ProfileWim JH Hagen, View ORCID ProfileRobert A Dick, Lingbo Yu, Abhay Kotecha, View ORCID ProfileFlorian KM Schur
doi: https://doi.org/10.1101/2022.01.10.475481
Martin Obr
1Institute of Science and Technology Austria, Klosterneuburg, Austria
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Wim JH Hagen
2European Molecular Biology Laboratory (EMBL) Heidelberg, Germany
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Robert A Dick
3Department of Molecular Biology and Genetics, Cornell University, Ithaca, USA
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Lingbo Yu
4Materials and Structural Analysis Division, Thermo Fisher Scientific, Eindhoven, Netherlands
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Abhay Kotecha
4Materials and Structural Analysis Division, Thermo Fisher Scientific, Eindhoven, Netherlands
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Florian KM Schur
1Institute of Science and Technology Austria, Klosterneuburg, Austria
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  • For correspondence: florian.schur@ist.ac.at
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Abstract

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) image processing has been well documented for single particle analysis (SPA). 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. 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.

Competing Interest Statement

Abhay Kotecha and Lingbo Yu are employees of Thermo Fisher Scientific. The other authors declare no competing interests

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted January 11, 2022.
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Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs
Martin Obr, Wim JH Hagen, Robert A Dick, Lingbo Yu, Abhay Kotecha, Florian KM Schur
bioRxiv 2022.01.10.475481; doi: https://doi.org/10.1101/2022.01.10.475481
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Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs
Martin Obr, Wim JH Hagen, Robert A Dick, Lingbo Yu, Abhay Kotecha, Florian KM Schur
bioRxiv 2022.01.10.475481; doi: https://doi.org/10.1101/2022.01.10.475481

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