Following this thermodynamic cycle, firstly, the ligand located in solvent (state A') is decoupled from its environment (state B'). The decoupled ligand (state B') is allowed to freely sample the whole simulation box. To be able to proceed with the second leg of the cycle, i.e. coupling the ligand to the system in the protein's binding site, the ligand needs to be restrained to the protein (state B). The contribution of the added restraints is taken into account analytically . Finally, the ligand in the protein's binding site is coupled to the system and the restraints are removed (state A). The free energies for the two legs of the thermodynamic cycle are obtained separately by performing multiple non-equilibrium transitions in the ligand coupling and decoupling directions, recording the work distributions and using the Crook's Fluctuation Theorem 11 to evaluate the free energy differences.