11. 3-Iodotoluene binding to T4 lysozyme L99A Table . Binding free energy ∆G • for the 3-iodotoluene/T4 lysozyme system. We show the free energy difference of each individual leg of the thermodynamic cycle as well as the binding free energy calculated by summation along the cycle. Reported uncertainties are the standard deviation of three independent replicates. ∆G restraints of f was calculated analytically, using the formula in Boresch et al. Costs for restraining and releasing the restraints on Val111 were 0.1 and -0.1 kcal/mol. The cumulative free energy difference is plotted as a function of the number of transitions. We tested four different protocols that differ in the length of the non-equilibrium transition and the number of transitions. Three independent replicates (blue, orange, green) were run for each protocol. Uncertainties were estimated via bootstrapping. The different protocols did not converge to the same free energy difference in the simulation time. Figure . Free energy difference for releasing the dihedral restraints in the decoupled state of the 3-iodotoluene/ T4 lysozyme system in the protocol where multiple dihedrals were restrained. The alchemical path was nonlinear (blue and green curve) or linear (orange curve). For the EQ approach we show the mean free energy difference across six replicates while for the NEQ approach the free energy estimate was obtained by pooling work values from six replicates and estimating one free energy difference using BAR (Sec. 4.5). The uncertainty estimate in both approaches is the standard deviation across six replicates. All protocols converged to the same free energy difference within uncertainty. In the NEQ approach, a nonlinear alchemical path, where more sampling time can be spend in the more challenging part of the transformation (lower force constants) did not perform better than the protocol using a linear alchemical path. The standard deviation was high (0.6-1.1 kcal/mol) indicating sampling problems in this protocol where multiple additional side chains were restrained. Input topology files for the nonlinear NEQ protocol are provided in the SI files. For the EQ approach we show the mean free energy difference across three replicates while for the NEQ approach the free energy estimate was obtained by pooling work values from three replicates and estimating one free energy difference using BAR (Sec. 4.5). The uncertainty estimate in both approaches is the standard deviation across three replicates. Here we only show λ 8 for the three independent replicates, additional λ windows can be found in the SI. Sudden changes in dH/dλ correlated with the entry/escape of water molecules into the space between protein and ligand. Water sampling was inadequate which shows that introducing the bubble-ligand only separates the water sampling from the decoupling of the ligand, but does not solve the problem.