Streptavidin. Streptavidin is a bacterial protein that binds to biotin with femtomolar affinity 162 , among the highest known. Streptavidin is a dimer which binds one biotin molecule with each monomer, and since the availability of the first experimentally determined structures of the complex 163 , their interaction has been extensively investigated . Multiple hypotheses have been made for the factors responsible for the exceptional affinity. The binding site is prevalently hydrophobic 117 , but the ligand establishes both van der Waals and hydrogen bond interactions . Since the cumulative effect of these direct interactions is not sufficient to account for the measured affinity, cooperative hydrogen bonds and multiple hydration shells have been considered . MD simulations identified a conserved pattern of water molecules in the ligand binding site , forming a five-membered ringlike structure which occupies the position of of the bound ligand bi-cyclic core. Multiple studies suggested that those hydration sites are geometrically restricted, leading to unfavorable translational and orientational entropy. Consequently, their displacement is expected to play an important role in biotin binding. These characteristics make this the streptavidin-biotin system an interesting target for testing the performance of WaterKit. Beside interactions with themselves, water molecules composing the ring are stabilized by a precise network of hydrogen bonds with polar side chains of Thr78, Asp116, Asn11, Ser15 and Ser33 surrounding the ring and nearly coplanar with it. Above and below the ring, the highly structured water cluster is capped by hydrophobic side chains of Trp96 and Leu13 (below the plane) and Leu25, Val47, Trp108, and Leu110 around and above the ring. The analysis of hydration sites predicted with the MD simulations and WaterKit (Fig. 10) shows that the five-membered structure is apparent in the oxygen densities calculated with both methods, but WaterKit is not able to correctly place two of them (W2 and W5) within 1 Å from the MD positions. For W2, WaterKit predicts two distinct hydration sites at 1.3 and 1.5 Å from the MD position, placing them at the edges of the MD-predicted oxygen high density location (gO map, Fig. ), with the closest water overlapping with it. Hydration site W1 matches very closely two water molecules found in a recent high-resolution apo structure of streptavidin (Fig. ). For W5, on the other hand, both MD and WaterKit show a more marked density smear, resulting in WaterKit placing the water at 1.5 Å from the MD position. Other weakly connected water molecules are predicted by both methods overlapping with the aliphatic tail of the biotin pentanoic acid, loosely matching water molecules found in various apo structures of streptavidin (Fig. ). Finally, both methods predict hydration sites, WA and WB, located at the positions occupied by the carboxylic oxygen atoms of the acid, and which are visible in these apo structures, but are displaced upon ligand binding (Fig. ). While a certain degree of variability is found between Wa- terKit and MD predictions (especially on more unstable water positions), both methods capture the overall features of the site, including the key five-membered ring hydration site.