Structure of dethiobiotin synthetase at 0.97 angstrom resolution

The crystal structure of the 224-residue protein dethiobiotin synthetase fr om Escherichia coli has been refined using X-ray diffraction data at 0.97 A ngstrom resolution at 100 K. The model, consisting of 4143 protein atoms in cluding 1859 H atoms and 436 solvent sites, was refined to a final R factor of 11.6% for all reflections, and has an estimated mean standard uncertain ty for the atomic positions of 0.022 Angstrom, derived from inversion of th e blocked matrix. The structure was refined with a full anisotropic model f or the atomic displacement parameters using SHELX97. Stereochemical restrai nts were applied throughout the refinement. In the last cycles, the planari ty of the peptide bonds was not restrained, resulting in a mean omega value of 179.6 degrees. Analysis of the most anisotropic regions of the protein shows that they form four clusters of residues. Alternate conformations for the side chains of 15 residues and for the main-chain atoms of six residue s from three loops were included in the model. An analysis of C-H ... O hyd rogen bonds shows that such interactions occur rather frequently in DTBS; i n total, 16 such hydrogen bonds were found. In the central beta-sheet, 13 C -H ... O bonds between carbonyl O and C alpha H atoms were found. Other int eractions of this type involve main-chain-side-chain and side-chain-sidecha in C-H ... O bonds. The model includes 436 water sites, of which 233 molecu les form the first hydration shell. Analysis of the protein-solvent interac tions shows that about one third of the accessible surface of the enzyme is not covered by ordered solvent. No difference in propensity for ordered so lvent close to hydrophilic or hydrophobic surface areas was found. The comp arison of the 100 K structure with the structure of the enzyme determined a t room temperature shows several regions with different conformation, inclu ding areas in the active site, suggesting that structural transitions can o ccur during flash freezing. This observation questions one of the basic ass umptions in the analysis of enzymatic reaction mechanisms using cryocrystal lography.