The crystal structures of unligated adenylosuccinate synthetase from Esherichia coli in the space groups P21 and P212121 have been refined to R-factors of 0.199 and 0.206 against 2.0 and 2.5A data, respectively. The dominant structural motif of the synthetase monomer is a central 10 stranded [beta]-sheet composed of 9 parallel strands and a 10th antiparallel strand. Four subdomains extend off of this [beta]-sheet and contribute loops of residues which are disordered. The dimer interface involves two nearly-independent regions. A pair of helicea (H4 & H5) interacting with their symmetry equivalent mates defines the first region. The second region involves structural elements which connect strands of the central [beta]-sheet. Residues in the putative IMP binding site are also located at or near the dimer interface;The P212121 crystal form allows gradual infusion of guanosine triphosphate ribonucleotides. Structure of the synthetase complexed with guanosine-5'-([beta], [gamma]-imido)triphosphate and guanosine-5'-([beta],[gamma]-methylene)triphosphate in the presence and absence of Mg2+ have been refined to R-factors below 0.20 against data to 2.7A. Asp333 of the synthetase hydrogen bonds to the endo- and exocyclic amino groups of the guanine base moiety. The hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. Lys331 and Pro417 are involved in hydrophobic contact with either face of the guanine base. The synthetase does not recognize the N7 position of the guanine base or the ribose or the polyphosphate group. Even though Mg2+ was present in the soaking solution, no electron density was observed for Mg2+. These structures represent an initial encounter complex;Structures of the synthetase complexed with Mg2+, IMP, GDP, NOsp3-, and hadacidin at 298 and 100°K have been refined to R-factor below 0.20 against data to 2.7 and 2.5A resolution, respectively. There is a large (9A) conformational change in the loop from 42-53 which flanks the GDP binding site. Loops 120-130 and 299-303 are ordered in these structures. Peptide linkage between residues 40-41 and 223-224 reorient so that the main chain carbonyl of Ala40 can coordinate to the Mg2+ and the amide of Gln224 can hydrogen bond to the NOsp3-.;Mg2+ is five coordinated with the possibility of a sixth coordinate bond to OD1 of Asp13. Oxygen atoms from GDP, NOsp3- and hadacidin define the equatorial plane of coordination. The apical ligand of the Mg2+ is the carbonyl of Ala40. NOsp3- is recognized by two interactions at each of its three oxygen atoms. His41 may form a bifurcated hydrogen bond to one of the oxygens on the [beta]-phosphate of GDP and to one of the oxygen of the NOsp3-. Hadacidin is recognized at its [beta]-carboxylate by the main chain amides of residues 299-303, as well as by the side chain of Arg303. The aldehyde group on hadacidin is coordinated to the Mg2+. IMP makes interactions at its 5'-phosphate with Asn38, Thr129, Thr239, and Arg147 from a monomer related by twofold symmetry. The base of IMP hydrogen bonds to side chains of Gln224 and Asp13 through its N7 and N1 positions, respectively.