Structure of dethiobiotin synthetase at 0.97 Å resolution

Abstract

The crystal structure of the 224-residue protein dethiobiotin synthetase from Escherichia coli has been re®ned using X-ray diffraction data at 0.97 A Ê resolution at 100 K. The model, consisting of 4143 protein atoms including 1859 H atoms and 436 solvent sites, was re®ned to a ®nal R factor of 11.6% for all re¯ections, and has an estimated mean standard uncertainty for the atomic positions of 0.022 A Ê , derived from inversion of the blocked matrix. The structure was re®ned with a full anisotropic model for the atomic displacement parameters using SHELX97. Stereochemical restraints were applied throughout the re®nement. In the last cycles, the planarity of the peptide bonds was not restrained, resulting in a mean 3 value of 179.6. 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 residues from three loops were included in the model. An analysis of CÐHÁ Á ÁO hydrogen bonds shows that such interactions occur rather frequently in DTBS; in total, 16 such hydrogen bonds were found. In the central -sheet, 13 CÐHÁ Á ÁO bonds between carbonyl O and C H atoms were found. Other interactions of this type involve main-chain±side-chain and side-chain±side-chain CÐHÁ Á ÁO bonds. The model includes 436 water sites, of which 233 molecules form the ®rst hydration shell. Analysis of the protein±solvent interactions shows that about one third of the accessible surface of the enzyme is not covered by ordered solvent. No difference in propensity for ordered solvent close to hydrophilic or hydrophobic surface areas was found. The comparison of the 100 K structure with the structure of the enzyme determined at room temperature shows several regions with different conformation, including areas in the active site, suggesting that structural transitions can occur during¯ash freezing. This observation questions one of the basic assumptions in the analysis of enzymatic reaction mechanisms using cryocrystallography.

Spatial distribution of radiation damage to crystalline proteins at 25–300 K

“…An understanding of these motions can be obtained through their temperature dependence (Frauenfelder et al, 1979(Frauenfelder et al, , 1991Tilton et al, 1992;Chong et al, 2001) and temperature can be used as a tool to trap intermediates in kinetic crystallography (Bourgeois & Royant, 2005;Colletier et al, 2007Colletier et al, , 2008Bourgeois & Weik, 2009;Weik & Colletier, 2010). Low-temperature (100 K) structures often miss functionally important information (Deacon et al, 1997;Karplus et al, 1997;Sandalova et al, 1999;Scheidig et al, 1999). 'Hidden' (i.e.…”

Biomolecular cryocrystallography: Structural changes during flash-cooling

“…This fact is probably widely appreciated within the crystallography community, although rarely stated explicitly. With a few notable exceptions (6)(7)(8)(9)(10)(11)(12), it is usually taken for granted that the cryostructure is quenched at ambient temperature (T* ϭ T 0 in our notation) and, therefore, that the only structural effect of the low temperature is to sharpen the atomic displacement distributions without significantly displacing the mean atomic positions [apart from a more-or-less uniform contraction of the protein (6-9)]. In contrast to this conventional wisdom, the present analysis indicates that many degrees of freedom are quenched at temperatures near 200 K, where local conformational and association equilibria may be strongly shifted toward low-enthalpy states.…”

“…Whatever the cause, these findings raise concerns about the validity of cryostructures of biomolecular complexes (9). Significant conformational differences between cryogenic and room-temperature protein structures have been reported also for solvent-exposed side chains (not located at crystal contacts) and associated water molecules (10)(11)(12), sometimes in the catalytic site of enzymes.…”

Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation

“…The BioD reaction is the ATP-dependent formation of dethiobiotin from DAPA and CO 2 . The enzyme is a homodimeric protein (subunit of 24.1 kDa) that is structured into a single well folded domain (45)(46)(47)(48). X-ray crystallographic studies have shown that the reaction proceeds by carbamoylation of N-7 of DAPA (45,46) (Fig.…”