Proton-transfer pathway including the water network and His64. The entrance-conduit waters (WEC) are coloured cyan and the intermediate waters (WI and WI′) are coloured steel blue for clarity. The electron density (2Fo − Fc) is contoured at 1.5σ where not indicated otherwise. The major hydrogen bonds between water molecules are represented by dashed lines, while alternative hydrogen bonds that are mutually exclusive are represented by dotted lines. (a)–(d) Native CA II structures. (e)–(h) V143I CA II structures. WI′ at 7, 13 and 15 atm and W′EC1 at 15 and 13 atm are contoured at 1.25σ, and WI at 0 atm is contoured at 1.0σ. The inset (red box) in V143I-0atm shows the difference map (Fo − Fc contoured at 3.0σ; green) when the HCO3− molecule is not included in the structure refinement.