For the cases where multiple experimentally resolved structures are available, it may not be evident which structure would be best suited for initializing simulations to obtain a representative apo state ensemble. It is, however, possible to exploit binding free energy calculations to identify the structure yielding the most probable conformational ensemble. This analysis does not require any knowledge of the actual (experimentally measured) set of binding affinities. It rather relies on multiple calculations of the binding affinities connecting one holo struc-ture with multiple possible apo states (Fig ). We use phosphodiesterase 2 (pde2) complexed with 21 inhibitors to illustrate this approach. Numerous experimentally resolved monomeric pde2 structures are available, where the protein is crystallized in its apo state (e.g. 4htz ) or in a complex with a ligand (e.g. 6ezf , 4d08, 4d09 8 ). Availability of these structures allows constructing a set of apo states by using either an actual apo conformer from the crystallographic structure or by removing a ligand from a holo structure. In principle, the most likely apo state is at its free energy minimum, i.e. of the multiple candidate conformers, the one with the lowest free energy would be the most populated in the ensemble. However, calculating free energy differences between the apo conformers directly is a computationally highly demanding challenge.