Table . Binding free energy ∆G • for the HSP90 system using the bubble approach. 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. The cumulative free energy difference is plotted against the number of transitions. Transitions were run for 1 ns. The standard deviation across three independent replicates was 0.4 kcal/mol which is higher than in the protocol with stronger force constant on the restraints (0.2 kcal/mol). (b) Work values for the ligand binding to HSP90. Shown are values for replicate 1 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 similar in both protocols, in the reverse direction 18.7 kcal/mol for the protocol with a weaker force constant and 15.2 kcal/mol for the original protocol, and in the forward direction 11.5 vs. 13.9 kcal/mol.