We also tested a different protocol, where we restrained the dihedral to the orientation in the holo structure both in the bound and unbound state and did not switch it upon decoupling of the ligand. This introduced new sampling problems: Other side chains in the binding site (methionine 102, valine 103, isoleucine 78) reoriented in response to the forced unfavorable orientation of Val111 in the unbound state (probably to fill the space which would otherwise be empty) making it necessary to restrain those as well. The Pearson correlation coefficient between the dihedral angles and the work values helped detect some of these DOF. This metric was however sometimes subject to noise since many dihedral angles correlated with work values. Correlations were also not always consistent across replicates because different side chain caused problems in different replicates. Restraining the dihedral angles of those side chains as well led to a low standard deviation (0.2 kcal/mol) in the NEQ approach already with short transitions (100 ps/transition).