The p38α protein-ligand complex shows a particularly strong dependence of the calculated ∆G on the starting structure. For this case, we were able to identify the particular structural details that are responsible for more than 9 kJ/mol offset in the calculated ∆G values One of the main differences between the apo (pdb id 1wfc ) and holo (3fly) structures occurring close to the binding site is a major loop motion: colored in orange and blue in Figure . However, it appears that even the short (10 ns) equilibrium simulations that we employed in the current protocol are sufficient to sample this loop transition (Fig ). We have also explicitly probed whether this structural feature may modulate the accuracy of the calculated ∆G values. We have filtered the starting structure ensemble for the ligand coupling transitions, retaining only those conformers with a loop position similar to the one from the crystallographic apo structure. This, however, had no effect on the calculated binding ∆G values (Fig ).