Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis

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.