This only occurred in the weakly interacting states of the ligand, but affected the free energy difference as shown in the correlation between dH/dλ values and the RMSD of the ligand (Figure ). The binding mode flip also had an impact in the NEQ approach as judged by a high PCC (PCC=0.61) between reverse work values and the ligand RMSD (SI Figure ). To prevent this conformational change in the ligand, one could change the ligand atoms involved in the orientational restraints to also include the ring that flipped in the binding site in order to stabilize the binding mode better in the weaklyto non-interacting alchemical region. Another option would be the use of RMSD restraints. Finally, minor problems were caused by side chain rearrangement of asparagine 36 (Asn36) upon binding. But since reorientation seemed not to be directly correlated with dH/dλ values nor to the work values in the NEQ approach, the impact of this DOF on the estimate of the free energy differences is not clear. However, this Asn36 rearrangement does have a Here we show λ 13 across all three replicates as an example; additional data is in the SI. The entry of water molecules (more than the three that were already present in the starting structure) was a slow motion and had an impact on the potential energy of the system. Equilibrium was not reached at this particular λ window in any of the three repeats. huge impact on the ∆G estimate in the "bubble approach" as described in Section 5.5.3. It is not clear whether the sampling problem here is overshadowed by more severe problems in water and binding mode sampling, or whether it is directly linked to the bubble approach.