Restraining all rotatable bonds in the ligands increased the efficiency of these simulations, suggesting that sampling of different rotamers might be slow in this system. The standard deviation of the protocols that used dihedral restraints was overall lower than in the two protocols where the rotatable bond was not restrained (Figure ). The thermodynamic cycle was adapted accordingly to account for the contribution of the restraints (Section 4.). Using an enhanced sampling protocol that softens the vdW parameters and performs HREX (Figure SepTop ϵ-HREX no dihre) improved the sampling of different rotamers over the standard protocol (SepTop EQ no dihre), shown by the smaller standard deviation across three independent repeats.