Table . Protein (P1, P2, P3) and ligand atoms (L1, L2, L3) involved in Boresch-style restraints. One distance (P3-L1), two angles (P2-P3-L1 and P3-L1-L2) and three dihedrals (P1-P2-P3-L1, P2-P3-L1-L2 and P3-L1-L2-L3) were restrained to their value in an equilibrated reference structure. Free energy difference for decoupling toluene in the binding site with the NEQ protocol using weaker Boreschstyle restraints. The cumulative free energy difference is plotted against the number of transitions. The standard deviation across three independent replicates is 0.2 kcal/mol which is slightly lower than in the protocol with higher force constant on the restraints (0.3 kcal/mol). (b) Work values for toluene binding to T4 lysozyme L99A. Shown are values measured for each attempted transition, in forward (blue) and reverse (orange) direction, as well as the distribution of the work values. The reverse work distribution is similar to the one in the protocol using stronger restraints and the mean dissipation was independent of the restraint strength and in both protocols 1.7 kcal/mol in the reverse direction and 0.9 kcal/mol in the forward direction.