We then focus our sampling on just binding sites, where we first establish whether GCNCMC can sample multiple binding modes correctly. Owing to the rigorous acceptance criteria for the GCNCMC moves, only the move proposals that generate a plausible configuration for a given concentration will be accepted. Through constant insertion and deletion move proposals, the resulting binding modes should be reproduced with appropriate populations. To confirm, we look at the binding modes in simulations of a model host-guest system (β-cyclodextrin) and for toluene binding to T4L99A. The latter has been shown to bind in two distinct orientations along with their symmetric equivalents, giving a total of four binding modes. Finally, we then exploit the concentration dependence in the acceptance criteria to apply a titration protocol (Fig. ) to calculate the binding affinities for small fragment molecules in the aforementioned systems. This method, unlike ABFE, does not require prior knowledge of binding modes, artificial restraints, and symmetry corrections. The results are compared to a more traditional ABFE calculation, which is often regarded as the gold standard in the field of computational free energy prediction. The protocols used are described in both the "Methods" section and the Supplementary Information.