Fig. 3: Setup-dependent parameters and calibration uncertainty.
a, The distribution of the parameters quantifying the statistics of the measurements and the performance of the setups used for both MalE and U2AF2 measurements are shown as histograms and violin plots for the measurements from eight laboratories. The circle and whiskers in the violin plot indicate the mean and standard deviation (n = 64, averaged over eight samples measured in the eight different laboratories). Sample-dependent distributions of the shown parameters are given in Supplementary Fig. 9. b,c, Pairwise plots of the average count rate (b) and the number of photons (c) against the burst duration. The same datasets are plotted as used for a. While the count rate decreases slightly for longer burst durations, a positive correlation is observed for the acquired number of photons per burst and the burst duration, indicating that larger observation volumes result in a higher accumulated signal per molecule. Correlations between all parameters are shown in Supplementary Fig. 10. Error bands indicate the 95% confidence intervals of the regression. d, The distributions of the four correction factors for the calculation of accurate FRET efficiencies for all the MalE measurements are shown as histograms and violin plots for the measurements from all laboratories. The circle and whiskers in the violin plot indicate the mean and standard deviation (n = 64, averaged over eight samples measured in the eight different laboratories). e, A plot of the standard deviation of the reported FRET efficiencies from 16 laboratories (as a measure of the experimental uncertainty) against the average FRET efficiency for the MalE mutants 1–3 reveals that lower uncertainties are observed for higher FRET efficiencies. The black line represents a fit of the estimated uncertainties under the assumption that the variations arise solely due to uncertainty in the γ factor (equation (1)). The inferred relative uncertainty of the γ factor is around 23%. Shaded areas indicate relative uncertainties of 5–50%. Error bars indicate 95% confidence intervals around the average value.
