Another challenge for the absolute binding free energy calculations is posed by the need to explicitly sample the apo state of the protein, i.e. the protein without the bound ligand. As this state may substantially differ from the ligand bound (holo) state, the simulation method needs to be capable of capturing the free energy differences between the protein conformers. Non-equilibrium (NEQ) free energy calculations present an elegant solution to this challenge. Such calculations determine the free energy difference by performing rapid out-of-equilibrium ligand coupling/decoupling transitions initialized from the equilibrium protein apo and holo ensembles (Fig ). This allows one to explicitly include the different apo and holo end-states into the same calculation . Several recent applications of the NEQ approach on model host-guest systems showed promising results for the calculation of absolute binding free energies .