Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.
Danial, John SH
Margheritis, Eleonora G
Chumpen Ramirez, Sabrina
J Phys Chem Lett
American Chemical Society (ACS)
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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
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.
Single Molecule Imaging
Deutsche Forschungsgemeinschaft (309966, INST 190/194-1, UN111/13-1)
European Research Council (309966)
External DOI: https://doi.org/10.1021/acs.jpclett.1c03835
This record's URL: https://www.repository.cam.ac.uk/handle/1810/334239
Attribution-NonCommercial-NoDerivatives 4.0 International
Licence URL: https://creativecommons.org/licenses/by-nc-nd/4.0/