3-Iodotoluene binding to T4 lysozyme mutant L99A. For 3-iodotoluene binding to T4 lysozyme (L99A), in both EQ and NEQ calculations the independent replicates did not converge to the same result in 400 ns total simulation time, indicating inadequate sampling. In Figure we show the mean and standard deviation for the independent repeats of the EQ, and different protocols of the NEQ approach; for details on the individual repeats see SI Figures and. The standard deviation σ for the EQ approach was higher than in the toluene case (σ 3-iodotoluene = 0.4 kcal/mol, σ toluene = 0.1 kcal/mol) suggesting that sampling problems were more severe. For the NEQ protocols, which all used the same total simulation time, the ∆G estimate depended on the length of the NEQ switching transitions. Since overlap of the work distributions was poor, even with 4 ns/transition (SI Figure ), results are likely not to be trusted. The overall binding free energy of 3-iodotoluene, which was obtained through the thermodynamic cycle, varies between -4.8 and -5.7 kcal/mol among different protocols, as shown in SI Table . The experimental binding affinity of 3-iodotoluene to T4 lysozyme L99A was not found in the literature. Here our focus is not on agreement with experimental values, since this is also a function of the force field and other factors; rather, we focus on whether we have obtained converged values. 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. Same amount of total simulation time was used for the EQ and the four protocols of the NEQ approach. The different NEQ protocols differ in the length and the number of NEQ transitions. The standard deviation, especially for the EQ approach, was high and the ∆G estimate of the NEQ protocols depended on the transition length, suggesting insufficiencies in sampling.