The C-type lectin domain of the rat mannose-binding protein contains a complex network of protein-carbohydrate-calcium-water interactions. The crystallographic geometry of the binding site was reproduced by molecular dynamics simulation. The integrity of the binding site geometry, however, is critically dependent on the presence of a third calcium ion in the mannose-binding protein. This ion is distant from the bound carbohydrate, and its presence was originally considered to be a crystallization artifact. A network of concerted motions spanning spatially distant secondary structure elements in the lectin domain was observed in the dynamical model only when the third calcium binding site was occupied. This cooperativity may be implicated in ligand binding stabilization.