While restraining the rotatable bond at different dihedral values in each window, we observed that sometimes it altered the binding mode of the ligand (Figure ). Ideally, we would like to sample only a single degree of freedom in umbrella sampling simulations -different orientations of the rotatable bond with the same configuration for the rest of the ligand in our case. Hence, we applied 250 kcal/mol/Å 2 positional restraint on the fixed part of the ligand (atoms outside the grey box in Figure ) during simu-lations with parameter settings (ii) and (iii) to ensure that the protein-ligand interactions are the same for the rest of the ligand.