PT  - JOURNAL ARTICLE
AU  - John S H Danial
AU  - Yuri Quintana
AU  - Uris Ros
AU  - Raed Shalaby
AU  - Eleonora G Margheritis
AU  - Sabrina Chumpen Ramirez
AU  - Christian Ungermann
AU  - Ana J Garcia-Saez
AU  - Katia Cosentino
TI  - Systematic assessment of the accuracy of subunit counting in biomolecular complexes using automated single molecule brightness analysis
AID  - 10.1101/2021.11.22.469513
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.11.22.469513
4099  - http://biorxiv.org/content/early/2021/11/22/2021.11.22.469513.short
4100  - http://biorxiv.org/content/early/2021/11/22/2021.11.22.469513.full
AB  - 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 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 stoichiometry analysis of such complexes.Competing Interest StatementThe authors have declared no competing interest.