These results provide further validation that all binding modes, and their symmetry equivalents, are sampled within a GCNCMC simulation. Consequently, this means that binding modes are inherently sampled in our titration calculations (Sec. 2.4), and thus are accounted for in the final free energy estimates, meaning there is no need for prior knowledge of the binding modes nor separate calculations for each mode. Fig. : Distribution of Toluene binding modes observed in GCNCMC/MD simulations. Dihedral angles between -π to -1.5 and 0 to 1.5 were assigned to binding modes A1 and A2 respectively. B1/B2 were assigned to angles between -1.5 to 0 and 1.5 to π. Inset: Pairwise RMSD between ligand poses projected onto PCA space and colored by the four clustered binding modes.