Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.
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Authors
Danial, John SH
Quintana, Yuri
Ros, Uris
Shalaby, Raed
Margheritis, Eleonora G
Chumpen Ramirez, Sabrina
Ungermann, Christian
Publication Date
2022-01-27Journal Title
J Phys Chem Lett
ISSN
1948-7185
Publisher
American Chemical Society (ACS)
Volume
13
Issue
3
Pages
822-829
Language
eng
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Danial, J. S., Quintana, Y., Ros, U., Shalaby, R., Margheritis, E. G., Chumpen Ramirez, S., Ungermann, C., et al. (2022). Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.. J Phys Chem Lett, 13 (3), 822-829. https://doi.org/10.1021/acs.jpclett.1c03835
Abstract
Analysis of single-molecule brightness allows subunit counting of high-order oligomeric biomolecular complexes. Although the theory behind the method has been extensively assessed, systematic analysis of the experimental conditions required to accurately quantify the stoichiometry of biological complexes remains challenging. In this work, we develop a high-throughput, automated computational pipeline for single-molecule brightness analysis that requires minimal human input. We use this strategy to systematically quantify the accuracy of counting under a wide range of experimental conditions in simulated ground-truth data and then validate its use on experimentally obtained data. Our approach defines a set of conditions under which subunit counting by brightness analysis is designed to work optimally and helps in establishing the experimental limits in quantifying the number of subunits in a complex of interest. Finally, we combine these features into a powerful, yet simple, software that can be easily used for the analysis of the stoichiometry of such complexes.
Keywords
Single Molecule Imaging
Sponsorship
Deutsche Forschungsgemeinschaft (309966, INST 190/194-1, UN111/13-1)
European Research Council (309966)
Identifiers
PMC8802318, 35044771
External DOI: https://doi.org/10.1021/acs.jpclett.1c03835
This record's URL: https://www.repository.cam.ac.uk/handle/1810/334239
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International
Licence URL: https://creativecommons.org/licenses/by-nc-nd/4.0/
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