Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex.
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The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data.The analysis indicates that at least 10 GFPAtCESA3 molecules can exist in each particle.These procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.
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Affiliation: Department of Biomedical Engineering, Huck Institutes of the Life Sciences, University Park, PA 16802 Interdisciplinary Graduate Degree Program in Cell and Developmental Biology, Huck Institutes of the Life Sciences, University Park, PA 16802.
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Mentions: Density estimation by a Gaussian mixture model (GMM) can provide predictions of peak position for each mode in a way that avoids the drawbacks of KDE. In this method, the distribution of steps is estimated by a mixture of Gaussians, and the means and variances of these Gaussians are obtained by maximizing the expected posterior probability, computationally achieved by expectation–maximization algorithms (Dempster et al., 1977). However, one uncertainty of this method is in choosing the number of Gaussians, k, to be fitted to the data, which can alter the fitting results. To provide an objective method for choosing the number of Gaussians, we fitted the step amplitude data using the Gaussian mixture model by an increasing number of Gaussians and determined the BIC value associated with each fit. The optimal number of Gaussians was defined as the number that gave the lowest BIC value, which for the simulated photobleaching data was 5 (Figure 6, A and B). The different peaks were assumed to be multiples of the unitary photobleaching amplitude, and the mean unitary step size was calculated as a weighted average of each peak, giving a value of 528.3 a.u. This estimate is within 6% of the step size value of 500 a.u. that was chosen for these simulated photobleaching data. 
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Affiliation: Department of Biomedical Engineering, Huck Institutes of the Life Sciences, University Park, PA 16802 Interdisciplinary Graduate Degree Program in Cell and Developmental Biology, Huck Institutes of the Life Sciences, University Park, PA 16802.