With this protocol, the work distributions in the NEQ case overlapped well already using 500 ps/transition (SI Figure ), indicating that indeed the Val111 side chain sampling led to poor overlap in the unrestrained calculations. However, a lot of NEQ transitions (∼ 320, SI Figure ) were necessary in order to converge independent replicates which was probably due to additional slow DOF in the system (perhaps Ile78 rotamer sampling). The approach of switching the dihedral during the alchemical path was challenging using the EQ protocol. The standard deviation across replicates was higher (std=0.4 kcal/mol, SI Figure ) than in the NEQ approach (std=0.07 kcal/mol) caused by insufficient sampling of the Val111 switching in some intermediate states.