Fig. 5: Assessing the accuracy of smFRET-derived distances in MalE.
a–d, AV calculations and model-based interdye distances. a, Schematic of Alexa546 attached to MalE (PDB 1OMP) showing the parameters needed for the AV calculations using the AV3 model6 (Supplementary Table 10). b, Fluorescence anisotropy decays of single-cysteine mutants for the donor (Alexa546, left) and acceptor (Alexa647, right) at the labeling positions K29C and S352C. Solid lines represent fits to a model with two or three rotational components (Supplementary Tables 11 and 12 and Supplementary Note 8). c, AV (light color) and ACV (dark color) calculations for Alexa546 (cyan) and Alexa647 (pink) at labeling positions 352 and 29. The zoom-ins show the mean positions of the dyes based on the AV (light shade) and ACV (darker shade) models. d, Comparison of the experimentally obtained FRET-averaged distance \(R_{\left\langle E \right\rangle }\) with the theoretical model distances using the AV (filled squares) and ACV (empty squares) calculations. Errors represent the standard deviation in experimental distances (n = 16 laboratories for MalE mutants 1–3, n = 2 laboratories for MalE mutants 4–5, n = 7 laboratories for U2AF). The solid line represents a 1:1 relation and the gray area indicates an uncertainty of ±3 Å for a Förster radius of R0 = 65 Å. MalE-4 and -5 were measured by two laboratories. e, Detection of dye-specific protein interactions. Top shows the five MalE mutants and U2AF2 labeled with different dye combinations to determine the donor–acceptor-combined residual anisotropy, 〈rc,∞〉tr,ss (n = 3 laboratories). Bottom shows the distance uncertainty relating to κ2, \({{\Delta }}R_{{{{\mathrm{app}}}}}\left( {\kappa ^2} \right)\), estimated (Supplementary Note 8). A maximum allowed distance uncertainty of ≤10% (shaded gray region) in \({{\Delta }}R_{{{{\mathrm{app}}}}}\left( {\kappa ^2} \right)\) leads to a dye-independent threshold of 0.25 for 〈rc,∞〉. f, The apparent dynamic shift 〈ds〉 versus the combined residual anisotropy 〈rc,∞〉 is shown for all measured dye pairs (top left) and individually. Error bars of the apparent ds represent the standard deviation over n = 3 laboratories. For the combined residual anisotropy, the propagated 1σ uncertainty (Supplementary Note 8). g, The structural flexibility of MalE estimated after filtering using the distance uncertainty threshold shown in e (Supplementary Note 12). Error bars represent the 1σ percentiles averaged over all dye pairs (n = 1, MalE-1; n = 7, MalE-2 and MalE-3; n = 4, MalE-4 and n = 5, MalE-5). The residual distance fluctuations obtained from control measurements on dsDNA in one laboratory (dsdsDNA = 0.0026 ± 0.0044) are shown as a black line (gray areas represent confidence intervals of 1σ, 2σ and 3σ).
