Baited reconstruction with 2D template matching for high-resolution structure determination in vitro and in vivo without template bias.

Autor:	Lucas BA; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United States.; Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, United States.; Center for Computational Biology, University of California Berkeley, Berkeley, United States., Himes BA; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United States.; Howard Hughes Medical Institute, Chevy Chase, United States., Grigorieff N; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United States.; Howard Hughes Medical Institute, Chevy Chase, United States.
Jazyk:	angličtina
Zdroj:	ELife [Elife] 2023 Nov 27; Vol. 12. Date of Electronic Publication: 2023 Nov 27.
DOI:	10.7554/eLife.90486
Abstrakt:	Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling. Competing Interests: BL, BH These authors are listed as inventors on a closely related patent application named "Methods and Systems for Imaging Interactions Between Particles and Fragments", filed on behalf of the University of Massachusetts. The patent relates to the use of the 2DTM method described in this manuscript, to image ligands and drugs bound to larger complexes that can be detected by 2DTM, NG This author is also listed as an inventor on a closely related patent application named "Methods and Systems for Imaging Interactions Between Particles and Fragments", filed on behalf of the University of Massachusetts. The patent relates to the use of the 2DTM method described in this manuscript, to image ligands and drugs bound to larger complexes that can be detected by 2DTM. Reviewing editor, eLife (© 2023, Lucas et al.)
Databáze:	MEDLINE
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