Three-level modeling of SSB wrapping configurations. Schematics of SSB, wrapped ssDNA (blue), and the distance between wrapped ends, xSSB (black arrow; top panels). Each extension change data point Δx(F) in Figure 3A corresponds to a curve in the space of possible Nw and xSSB, according to Equation 5 (colored curves, bottom panels). The widths of the curves correspond to the error bars in Figure 3A. Selected data points from Figure 3A are displayed (purple: F = 0.8 pN, Δx = 11 nm, blue: 4 pN, 14 nm, green: 7 pN, 10 nm, and red: 9 pN, 7 nm). At the first level of modeling (left panels), xSSB is assumed to be limited only by the size of the protein (i.e., xSSB < 6.5 nm; dark gray shaded area). The range of possible Nw corresponding to each selected data point is shown by the colored dotted lines. At the second level (middle), the range of possible xSSB is refined by utilizing the (SSB)65 crystal structure. The end-to-end distance between every pair of nucleotides ni and nj along the ssDNA in the structural model defines a lower and upper bound of xSSB for each Nw (gray shaded area). This, in turn, narrows down the range of possible Nw for each data point (colored dashed lines). At the third level (right), four ‘hotspots’, residues on each SSB monomer with which nucleotides interact most strongly (green molecular surfaces in the schematic and green nucleotides), are used to refine the estimates for xSSB further. Three regions near the hotspots (black contours) are identified and used to calculate xSSB. The numbering (1, 2, and 3) corresponds to the configurations shown in Figure 3—figure supplement 2. This analysis provides the narrowest estimate for the range of Nw for each data point Δx (colored bands). The best estimates for Nw are obtained from the center of this range (black dots); these are plotted in Figure 3C vs force.