Figure 3.

Comparison of data from targeted Y2H studies and true homology modeling methods. (A) To assess the ability of true homology modeling methods to predict the probability of detecting E2/E3-RING complexes in Y2H assays, the predicted free-energy values for 3150 E2/E3-RING complexes were compared with experimental profiles detected in Y2H studies. A binary score (1/0 = interaction was/was not observed) was assigned for each complex tested in Y2H assays. SigmaPlot 10.0 (Systat) was then used to fit the set of free-energy/binary score data pairs to the logistic model: p(Y2H interaction | free-energy score = x) = a / (1 + exp (−(x + b) / c)), with values for the parameters a, b, and c obtained by nonlinear regression. The resulting regression curve is shown in black. Black vertical lines indicate the distribution of the predicted free-energy values for interacting and noninteracting pairs on the upper and lower horizontal axes, respectively. Predicted free-energy values were ordered and split into bins of 200, and the frequency of Y2H interactions within each bin is shown as a gray horizontal bar (bar width indicates the free-energy values covered by each bin). (B) Distribution of free-energy values for all predicted human E2/E3-RING complexes (open bars); “strong” Y2H interactions selected on −Ade and −His + 2.5 mM 3AT plates (black bars); “weaker” Y2H interactions selected on −His + 2.5 mM 3AT plates (dark gray bars); interactions tested but not detected in Y2H studies (light gray bars). Dashed line indicates the approximate predicted free-energy values for the structurally defined UBE2L3–CBL complex (−7.87 ΔG int kcal/mol).