Table . Comparison of the free energy difference for decoupling toluene in T4 lysozyme L99A using Boresch-style restraints and center-of-mass (COM) -COM flat-bottom harmonic distance restraints. For the COM-COM restraint protocol we first turned the Boresch-style restraints off while turning on a distance restraint between the COM of the ligand and the COM of the side chain alanine 99. Then we decoupled the ligand and afterwards switched the COM-COM restraint back to the Boresch-style restraints in the noninteracting state. Reported uncertainties are the standard deviation of three independent replicates. In the NEQ approach, the dissipated work was higher in the COM-COM restraint protocol (3.8 kcal/mol in the reverse direction and 1.4 in the forward direction) than in the protocol using Boresch-style restraints. With a single COM-COM distance restraint, the ligand can sample the sphere around the radius of that distance restraint which presumably caused the larger dissipation. Free energy difference for decoupling toluene in the binding site with the NEQ protocol using dihedral restraints. The cumulative free energy difference is plotted against the number of transitions. Three independent replicates seem to converge to the same free energy difference and the standard deviation was lower in this protocol compared to the unrestrained calculations (0.06 vs. 0.3 kcal/mol).(c) and (d) Analyzing the set of forward and reverse data of replicates separately. Three replicates give three sets of transitions in the forward direction and reverse direction which are independent from one another. We took a fixed set of transitions from one replicate for one direction (in (c) the set of transitions from the first replicate was used for the reverse direction (B2A 1)) and calculated the free energy difference using data from the other direction of all three replicates (in (c) A2B 1 in blue uses transitions in the forward direction from replicate one). The same was performed for the other direction in (d). The free energy difference of the second replicate (orange) is higher in the forward direction (c) and lower in the reverse direction (d). This shows that the apparent convergence (and low standard deviation) seen in (b) is likely a coincidence.