The solution structures of staphylococcal nuclease (nuclease) H124L and its ternary complex, (nuclease-H124L).pdTp.Ca2+, were determined by ab initio dynamic simulated annealing using 1925 NOE, 119 phi, 20 chi 1 and 112 hydrogen bond constraints for the free protein, and 2003 NOE, 118 phi, 20 chi 1 and 114 hydrogen bond constraints for the ternary complex. In both cases, the final structures display only small deviations from idealized covalent geometry. In structured regions, the overall root-mean-square deviations from mean atomic coordinates are 0.46 (+/- 0.05) A and 0.41 (+/- 0.05) A for the backbone heavy atoms of nuclease and its ternary complex, respectively. The backbone conformations of residues in the loop formed by Arg81-Gly86, which is adjacent to the active site, are more precisely defined in the ternary complex than in unligated nuclease. Also, the protein side chains that show NOEs and evidence for hydrogen bonds to pdTp (Arg35, Lys84, Tyr85, Arg87, Tyr113, and Tyr115) are better defined in the ternary complex. As has been observed previously in the X-ray structures of nuclease-WT, the binding of pdTp causes the backbone of Tyr113 to change from an extended to a left-handed alpha-helical conformation. The NMR structures reported here were compared with available X-ray structures: nuclease-H124L [Truckses et al. (1996) Protein Sci., 5, 1907-1916] and the ternary complex of wild-type staphylococcal nuclease [Loll and Lattman (1989) Proteins Struct. Funct. Genet., 5, 183-201]. Overall, the solution structures of nuclease-H124L are consistent with these crystal structures, but small differences were observed between the structures in the solution and crystal environments. These included differences in the conformations of certain side chains, a reduction in the extent of helix 1 in solution, and many fewer hydrogen bonds involving side chains in solution.