Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex.
Bottom Line: 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 GFP-AtCESA3 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.
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.Show MeSH
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Mentions: To validate their performance, we first tested the step detection algorithms on simulated stepping data having SNR values from 0.4 to 5 (Figure 3). The step times were sampled from an exponential distribution with an expected value of 100 time points/plateau, with 90% of steps being a unit step increase and 10% being a unit step decrease. At high SNR values, the mean predicted step size was close to the actual value, but with diminishing SNR, an additional peak corresponding to twice the unitary step size emerged (Figure 3A and Supplemental Figure S2). We defined two metrics, sensitivity and precision, to assess the performance of the algorithms. Sensitivity is defined as the proportion of the true steps that are identified by the step detection algorithm. Precision is defined as the proportion of identified steps that are true steps (see Materials and Methods). Overfitting will lead to high sensitivity and low precision (false positives), whereas underfitting results in high precision but low sensitivity (missed events). With SNR values >2, all four algorithms performed well and had both high-sensitivity and high-precision values (Figure 3, B and C). Reasonable predictions were obtained at SNR values between 1 and 2, but sensitivity and precision both fell sharply for SNR values <1. The BIC-based algorithms displayed a tradeoff between sensitivity and precision, with Bdetector1 (constant variance) having higher sensitivity and Bdetector2 (unequal variance) having higher precision (Figure 3, B and C, blue and green plots). In contrast, for the two-sample t test methods, Tdetector1 (assumed constant variance) and Tedector2 (assumed unequal variance) performed similarly (Figure 3, B and C, red and black plots).
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.