Fig. : The two binding modes of Toluene to T4L99A. The crystal pose, A1/A2, is shown in magenta (PDB: 4w53) and the meta-stable pose, B1/B2, is in cyan. The dihedral angle between the three Toluene atoms highlighted in yellow, and the CA atom of Arg119 is used to distinguish between the binding modes. Adapted from Gill et al. Using the dihedral angle identified by to distinguish between the bound configurations A and B, the populations obtained from GCNCMC simulations are shown in Figure . We observed a ratio of 67:33 between poses A and B, which is in remarkably good agreement with our free energy estimates and that of 65:35 reported by Gill et al. Reassuringly, we also observe population ratios of 33:34 between symmetry-equivalent poses A1/A2, and 17:15 between B1/B2, indicating thorough sampling. An interesting distinction between these results and those published by Gill et al. is that when using GCNCMC, we did not have to define a transformation between the poses (taken as center-of-mass rotation by Gill et al. when using BLUES ). Further, with GCNCMC, no prior knowledge of the multiple binding modes is required, as we have shown that these are naturally sampled by the method giving accurate populations.