Finally, now that we have validated the ability of GCNCMC, without prior knowledge, to predict binding sites and relative binding mode populations in a given site, we can apply our titration protocol to calculate the binding affinities of different fragment series to β-cyclodextrin, T4L99A, and MUP1. The structures of the fragments are shown in the Supplementary Information. Using a pre-calculated excess chemical potential for each ligand, as would be necessary for any free energy calculation, we simulate over a range of ligand concentrations via Adams value scanning (see Methods section). We then measure the average occupancy of the simulation at each value of B eq and fit a logistic function, Eq. 13, to these data. The fragment concentration for which the corresponding average occupancy is 50% is the dissociation constant, K D , which is easily related to the free energy of binding (Eq. 11).