Fig. 2: Absolute binding free energy thermodynamic cycle employed.

The free energy of binding, i.e. going from a ligand in solution (state a) to a protein-ligand complex (state e), is captured through a non-physical path. First, the electrostatics are annihilated to zero (state b) over 11 λ windows. This is followed by a further 21 λ windows which decouple the ligand van der Waals interaction from the solvent (state c). The decoupled ligand is then analytically restrained as defined by ref. ¹⁰² (state d). By accounting for this restraint the ligand state is then equivalent to a non-interacting ligand in a protein-ligand complex (state h). The ligand interactions with the environment are then turned back on, first re-coupling the van der Waals interactions over 21 λ windows (state g), followed by a further 11 λ windows to add back electrostatics (state f). Finally, the orientational restraints are turned off over 12 λ windows resulting in a fully interacting protein-ligand complex (state e).