The class IV adenylyl cyclase from Yersinia pestis has been engineered by site-specific mutagenesis to facilitate crystallization at neutral pH. The wild-type enzyme crystallized only below pH 5, consistent with the observation of a carboxyl-carboxylate H bond in a crystal contact in the refined structure 2FJT. Based on that unliganded structure at 1.9 A resolution, two different approaches were tested with the goal of producing a higher-pH crystal needed for inhibitor complexation and mechanistic studies. In one approach, Asp 19, which forms the growth-limiting dicarboxyl contact in wild-type triclinic crystals, was modified to Ala and Asn in hopes of relieving the acid-dependence of that crystal form. In the other approach, wild-type residues Met 18, Glu 25, and Asp 55 were (individually) changed to lysine to reduce the protein's excess negative charge in hopes of enabling growth of new, higher-pH forms. These 3 sites were selected based on their high solvent exposure and lack of intraprotein interactions. The D19A and D19N mutants had reduced solubility and did not crystallize. The other 3 mutants all crystallized, producing several new forms at neutral pH. One of these forms, with the D55K mutant, enabled a product complex at 1.6 A resolution, structure 3GHX. This structure shows why the new crystal form required the mutation in order to grow at neutral pH. This approach could be useful in other cases where excess negative charge inhibits the crystallization of low-pI proteins.