To support our findings that the large dissipation (SI Figure ) was caused by a side chain rearrangement upon binding and not by the Boresch-style restraints, we performed calculations applying a lower force constant on the orientational (Boresch-style) restraints (see SI Figure ). The average dissipated work W d in the forward and reverse directions was similar for the protocols using weaker and stronger restraints (3.7 vs. 3.9 kcal/mol in the reverse direction and 3.4 vs. 4.1 kcal/mol in the forward direction). These findings suggests that the Boresch-style restraints were not a major source for the dissipation in this system. Equilibrium FEC also suffered from sampling problems due to the rearrangement of the Val111 side chain. These issues were most severe in intermediate λ windows where the Val111 side chain showed partial occupancy of two rotamers and a slow rotation between them. In order to investigate this, we looked for sudden changes in dH/dλ values. Correlating these with the dihedral angle ξ1 of Val111 and comparing results among three independent replicates shows that the side chain was kinetically trapped in various intermediate states (Figure ). Approaches to overcome these sampling problems include the use of enhanced sampling techniques, like HREX or incorporating BLUES-like side chain moves. 57,75 5.3.3.