Finally, the overall binding free energy of toluene for both poses was obtained through the thermodynamic cycle (Table , results of individual legs of the cycle in SI Table ). A symmetry correction of -k B T ln 2 Figure . Decoupling of toluene in the binding site. The free energy difference for the EQ and NEQ approach for both poses is shown as a function of the sampling time. 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. The NEQ protocols differ in the lengths of the NEQ switching transitions, which are 200 ps, 500 ps and 1 ns, respectively. The number of switching transitions differs among the protocols in such a way that the same amount of overall sampling time is used in all protocols. All protocols converged to the same free energy difference within uncertainty. ≈ -0.4 kcal/mol was added to account for the symmetry-equivalent binding mode. The crystallographic pose was correctly predicted with a more negative ∆G with both methods (Table ). While the binding free energy for the non-crystallographic binding mode is not an experimental observable, when combined with the binding free energy for the crystallographic pose, it tells us the relative population of the two binding modes. ). The reorientation of the Ile78 dihedral angle occurred relatively fast on timescales of a few hundred ps -which is faster than simulation timescales and we seemed to be able to adequately sample the motion here (Figure and S3). This helped us develop techniques to better assess important protein motion. 5.2.2. Side chain reorientation in NEQ switching transitions. In the NEQ approach, end states have to be sampled long enough to start transitions from the correct distribution of dihedral angles and/or the transitions have to be slow enough to allow for reorientation (if needed) on ligand binding, otherwise the final distribution of dihedral angles will closely match the starting distribution.