The Coulomb interactions between the two ligands did not have to be excluded since in this thermodynamic cycle there are no end states where both ligands have electrostatic interactions turned on at the same time. As implemented in GROMACS, the Hamiltonian of an alchemical intermediate state is constructed by the linear interpolation of the Hamiltonians, rather than charges, i.e. H = (1 -λ) * H 0 + λ * H 1 , where λ is the alchemical parameter and H is the Hamiltonian of the alchemical state, H 0 the Hamiltonian of end state A, and H 1 the Hamiltonian of end state B. This means that even though both ligands may have partial electrostatic interactions at the same time, the ligands will not interact with one another at any state of the alchemical transition, as long as the ligands are not interacting with each other in the end states. The vdW interactions between the two ligand, however, have to be excluded since there are end states in the alchemical path (both end states in leg C Figure ) where both ligands have vdW partially or fully turned on.