Heat Shock Protein 90α (hs90a). The N-terminal domain of the Heat Shock Protein 90α (hsp90α, PDB 1uyg) is composed of 9 helices and an 8 strands antiparallel β-sheet that fold together into an α+β sandwich. The ligand binding pocket is located on the helical face at the center and it is widely open into the hydrophobic core of the structure. In it's open conformation 127 , a channel coming from the ATP binding site is present under this helix. Although mainly hydrophobic in nature, it contains numerous water molecules which play a key role in ligand binding, and have been exploited for inhibitors design . Four water molecules, named W1 to W4 in this study (Fig. ), are conserved in the same position in several crystallographic complexes 127,129-132 . These water molecules are part of a hydrogen bond network involving Asp93, Asn51, Ser52, Thr248 and Gly97. The role they play in ligand affinity and their displacement were studied in detail , making hsp90α also an ideal case for assessing the capabilities of the sampling method implemented in WaterKit. MD simulations identified 80 hydration sites found in at least a replicate, and 57 common in all triplicates. Of these sites, 44 and 27 are found also by WaterKit, respectively. Both MD simulations and Wa-terKit are able to identify the conserved water molecules W1-W4 (Fig. ) matching experimental coordinates very closely (< 0.5 Å), but their ranking differs. In MD simulations, W1 is correctly identified as the most stable (average E sw : -16.1 kcal/mol), while W2-W4 are considered roughly equivalent within 1 kcal/mol (E sw : -6.8, -6.1 and -5.8 kcal/mol, respectively). On the other hand, WaterKit ranks them as following: W1 < W4 < W2 ≈ W3, with E sw at -14.4, -10.2, -4.1 and -4.0 kcal/mol, respectively. Interestingly, the latter ranking is very close to what previously reported from a study based on IFST (a method analogous to the GIST approach used here ), which reported E sw for W1, W4, W3 and W2 to be -23.19, -16.11, -11.57 and -8.43 kcal/mol, respectively .