Fig. 2: Scatter plots showing the range of agreement of ΔGs between different experimental assays and FEP+ predictions.

Panels a–c compare measured affinities between two different experiments and panels d–f compare FEP+ predictions (y-axis) against experimental measurements (x-axis). Panels a and d show examples where the pairwise RMSE of relative binding free energies was much better than average, panels b and e show examples where the RMSE was close to the average, and panels c and f show examples where the RMSE was worse than average. The top left of each plot shows Kendall τ and pairwise RMSE for each data set. Points in the dark gray area are measurements or predictions that are within 1 kcal mol⁻¹ of each other, and points in the light gray area agree within 2 kcal mol⁻¹. a Shows that isothermal titration calorimetry (ITC) and fluorescence polarization (FP) binding free energy measurements of galectin ligands are offset by ~1 kcal mol⁻¹ - this offset is irrelevant for rank ordering and does not affect the correlation or the pairwise RMSE metric. The offset of the FEP+ ΔG predictions was determined by ensuring the mean of the ΔGs was equal to the mean of experimental ΔGs on the x-axis. Where the data was available, we included the reported standard error of the experimental measurements; the standard errors from FEP+ as calculated with the cycle-closure algorithm are also indicated on the bottom row of plots.