The I domain of the integrin alpha-L (ital) is about 175 residues long folded into a central β-sheet that is surrounded by α-helices, forming a Rossman fold. The crystallographic structure (pdb id: 2ica) shows that the binding site is widely open to the bulk solvent, but is mostly covered by non polar residues. Therefore, interactions between the ligand and the protein are mainly hydrophobic in nature except for the interaction of the urea carbonyl in the ligand with Glu284, Lys287 and Lys305 through a bridging water molecule. The overall results for ital show that WaterKit is predicting accurately ∆G with a r 2 of 0.92 and a RMSD of 1.25 kcal/mol against the MD average. In comparison, RMSD and r 2 between MD triplicates are about 0.37 kcal/mol and 0.98 for (Fig. , and Fig. and). In line with previous observations (Fig. ), a poor correlation is obtained for E ww term, despite a RMSD < 1 kcal/mol. Predictions were calculated over 33 hydration sites with an equivalent found in MD simulations. In total, 67 hydration sites were found in MD simulations, from which only 45 are common to all MD triplicates. Interestingly, WaterKit correctly predicted the position of water molecules in mostly hydrophobic regions, some of which appear to map on conserved features found in the ital ligand structures. In particular, hydration sites Wa, Wb and Wc are located in a fully hydrophobic part of the ligand binding pocket, and their positions overlap closely with the 1,3dichlorobenzene moiety (Fig. ), Their interactions with the protein are predicted to be weakly favorable by both methods (Fig. ) suggesting that they have been easily displaced by the ligand, possibly with an favorable entropy contribution the overall binding affinity. Water Wd, which is predicted to be involved in a water network involving the chrystallographic water W1 (W324 in the PDB structure) is predicted to be more stable, and it is displaced by one of the ligand carbonyl groups. In the case of the crystallographic water W1, it position in a bridging role was still accurately predicted with the simulations performed in absence of the ligand with both WaterKit and MD (Fig. ). The ∆G predicted with Wa-terKit (-6.5 kcal/mol) is smaller than the MD averages (-11.6 kcal/mol), possibly due to the higher entropy contribution estimates of the constrained simulation mentioned previously. Interestingly, other conserved water molecules predicted by Positive (TP) corresponds to the total number of water molecules correctly identified by WaterKit, using a distance cutoff of 1.0 Å. The different GIST energy components, Esw, Eww, TStrans., TSorient. as well as ∆G, were compared using the Root Mean Square Deviation (RMSD) and the coefficient of determination (r 2 ). RMSD and r 2 calculated for the MD triplicates is reported as a reference. (B) The water placement was evaluated using the True Positive Rate (TPR) (sensitivity) and the Positive Predictive Value (PPV) (precision) metrics. The sensitivity and precision were calculated on all the identified hydration sites, as well on different set of hydration sites based on their Esw energy from -5 to -2 kcal/mol. WaterKit but not by MD simulations coincide with ligand features such as the nitrogen of benzonitrile and the carboxythiophene groups.