Equilibrium MD Accepted GCNCMC Move Fig. : High level overview of the GCNCMC protocol. 1) Insertion and deletion moves occur within a user-defined region (grey sphere). 2) Moves are performed using a nonequilibrium switch occurring over a short time scale and are accepted or rejected according to the work done on the system over the move, W (X|Λ p ), the excess chemical potential of the molecule, µ ′ sol , the concentration of the molecule, c L , the number of molecules already in the region, N , and the volume of the defined GCMC region, V GCM C . 3) The resulting simulation is a regular MD simulation with GCNCMC moves interspersed. If a move is rejected (red dashed lines) the simulation restarts from the state prior to the move. 4) Binding affinities may be calculated by titrating the Adams value, B eq , and thereby concentration. More details can be found in the "Methods" section.