Instead, we can evaluate relative free energies of these conformers by connecting them via a common holo state. We calculate binding affinities for a set of 21 pde2 inhibitors using the structure 6ezf representing the protein-ligand complex and each of the 6ezf, 4htz, 4d08 and 4d09 structures independently representing the apo state. In this way we relate each apo state to one another via a common reference 6ezf holo state. Setting the free energy of the reference to 0 kJ/mol for convenience allows us to directly compare the apo states (Fig 5 ): the ∆G for an apo state is represented by averaged binding free energies calculated over the whole ligand set. The barrier (denoted with the dashed lines in Fig 5 ) is not attainable with this approach, as alchemical calculations do not explicitly probe the binding-unbinding pathway.