Here we keep the functional form of the hydrophobicity interaction unchanged and use a different reweighted ensemble as the target for further optimizing the parameters θ. Because the ensemble Z 1 agrees with the experimental data better than the original ensemble Z 0 , perturbation needed to reweight the ensemble Z 1 to match the experimental data should be smaller than that needed for the ensemble Z 0 . In other words, the reweighted ensemble as the target to further optimize the hydrophobicity parameters θ, which is equivalent to running a second iteration of the proposed framework in which the parameter θ 1 is used as the starting point.