Dataset. To design the method and assess its performance, we compiled a dataset of 10 proteins from the DUDe dataset , representative of different target topographies (deep vs. shallow) and topologies (solvent-accessible vs. closed). For nine of them, the PDB entries in DUD-e dataset were used: coagulation factor X (fa10, PDB id: 3kl6 ), FK506-binding protein 1A (fkb1a, PDB id: 1j4h ), heat shock protein HSP 90-α (hsp90α, PDB id: 1uyg ), leukocyte adhesion glycoprotein LFA-1 α (ital, PDB id: 2ica ), fatty acid binding adipocyte (fabp4, PDB id: 2nnq ), neuraminidase (nram, PDB id: 1b9v ), thymidine kinase (kith, PDB id: 2b8t 89 ) and cyclooxygenase-2 (pgh2, PDB id: 3ln1 ). For HIV-1 protease (hivpr), the structure used in the DUD-e (PDB id 1xl2 ) is characterized by an asymmetric conformation of the flap loops induced by the binding of an atypical ligand , which prevents the binding of the key structural water 301. Therefore this structure was replaced with 1hpx 92 and 2zye , both binding with the same potent inhibitor KNI-272. These structures were used to assess the impact of the backbone amide geometry of ILE50 and ILE50' on the water 301 position. Finally, the widely studied streptavidin-biotin complex (PDB id: 1stp ) was also added to perform a comparison with results reported for other desolvation estimate methods, such as the WaterMap method . Structure preparation. The preparation of the target structures for both methods was done using AmberTools19 toolkit . Crystallographic water molecules and Amber nonstandard residues were removed from the receptor, while ions were kept. Hydrogens were added using LEaP and N-and C-terminal charge patches applied according to the standard Amber protocol . Histidines were modeled in the default neutral form (HID) with one hydrogen atom on δ nitrogen. For hivpr, residue Asp25 of chain B was modeled in the neutral form (ASH) . Disulfide bridges were automatically detected during the preparation using pdb4amber () and built by LEaP. In order to use AutoGrid4 to calculate the grid maps, parmed () was used to convert output parameter, topology, and coordinate files, to the PDBQT format retaining Amber atom types and partial charges previously defined. Unlike the standard AutoDock protocol , an allatoms model is used and non-polar hydrogen atoms were kept (i.e., instead of being merged with heavy atoms).