ferent rotamers by scaling the LJ-ϵ down by multiplying it by a scaling factor of γ = 0.01. Free energies for transitions in the two opposite directions now converged to the same result (Figure ). Only the LJ-ϵ of atoms in the ethyl methyl ether group (Figure ) were scaled down by a factor of γ = 0.01, while for the rest of the molecule a scaling factor of γ = 0.5 was used. We had tried to scale down the LJ-ϵ of the entire molecule, however, found that this led to instabilities and convergence problems. We also found that convergence could be apparently achieved by restraining the dihedral of that rotatable bond during the simulation and releasing the restraints afterward. This however then led to higher standard deviations and slow convergence when releasing the dihedral restraint in the solvent. In the solvent, different rotamers were sampled in the interacting state which led to poor overlap of λ windows when releasing the dihedral restraint (results not shown). Overall, in this case the protocol which scaled back interactions of the mutated R-group performed best.