In a more quantitative study of multiple binding modes, we look at toluene binding to T4L99A, which has previously published simulation data from Gill et al. In that study, two toluene binding poses were identified: the crystal pose (denoted A1/A2) and a secondary pose (B1/B2). Our own free energy calculations of both poses revealed a free energy difference of 0.73 k B T , which translates to a population ratio of 68:32 at 298 K for poses A and B, respectively (Fig. ). This is in line with the ratio of 65:35 at 300 K reported by Gill et al. Note that A1/A2 corresponds to the symmetrically equivalent poses of A and likewise for B, owing to the C 2 symmetry axis of toluene.