Biomedical subjects

A Urzhumtsev

Publications and source records attributed to A Urzhumtsev.

7 recordsLinked to original sources

PubMed2003

On a fast calculation of structure factors at a subatomic resolution.

In the last decade, the progress of protein crystallography allowed several protein structures to be solved at a resolution higher than 0.9 A. Such studies provide researchers with important new information reflecting very fine structural details. The signal from these details is very weak with respect to that corresponding to the whole structure. Its analysis requires high-quality data, which previously were available only for crystals of small molecules, and a high accuracy of calculations. The calculation of structure factors using direct formulae, traditional for 'small-molecule' crystallography, allows a relatively simple accuracy control. For macromolecular crystals, diffraction data sets at a subatomic resolution contain hundreds of thousands of reflections, and the number of parameters used to describe the corresponding models may reach the same order. Therefore, the direct way of calculating structure factors becomes very time expensive when applied to large molecules. These problems of high accuracy and computational efficiency require a re-examination of computer tools and algorithms. The calculation of model structure factors through an intermediate generation of an electron density [Sayre (1951). Acta Cryst. 4, 362-367; Ten Eyck (1977). Acta Cryst. A33, 486-492] may be much more computationally efficient, but contains some parameters (grid step, 'effective' atom radii etc.) whose influence on the accuracy of the calculation is not straightforward. At the same time, the choice of parameters within safety margins that largely ensure a sufficient accuracy may result in a significant loss of the CPU time, making it close to the time for the direct-formulae calculations. The impact of the different parameters on the computer efficiency of structure-factor calculation is studied. It is shown that an appropriate choice of these parameters allows the structure factors to be obtained with a high accuracy and in a significantly shorter time than that required when using the direct formulae. Practical algorithms for the optimal choice of the parameters are suggested.

Journal Article↗

PubMed2001

Low-resolution data analysis for low-density lipoprotein particle.

The knowledge of the molecular structure of LDL, a large lipoprotein complex, is of great interest for medical investigations. Currently available LDL crystals do not diffract to high resolution and do not allow the application of standard crystallographic techniques. Additional difficulties arise because of a very dense crystal packing and the presence of several components with quite different mean densities. Several ab initio phasing methods previously reported by the authors have been successfully applied to find a crystallographic image of LDL at a resolution of 27 A. The most promising results have been obtained using direct phasing with a connectivity analysis of the electron-density maps. The current image makes it possible to discern a single particle covered by a layer of relatively high density that is asymmetrically distributed on the particle surface. It shows a partition of high and low densities inside the particle and, in particular, strips of varying density in the lipid core.

Crystallography, X-Ray↗

PubMed2000

Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center.

DNA-gyrase exhibits an unusual ATP-binding site that is formed as a result of gyrase B subunit dimerization, a structural transition that is also essential for DNA capture during the topoisomerization cycle. Previous structural studies on Escherichia coli DNA-gyrase B revealed that dimerization is the result of a polypeptidic exchange involving the N-terminal 14 amino acids. To provide experimental data that dimerization is critical for ATPase activity and enzyme turnover, we generated mutants with reduced dimerization by mutating the two most conserved residues of the GyrB N-terminal arm (Tyr-5 and Ile-10 residues). Our data demonstrate that the hydrophobic Ile-10 residue plays an important role in enzyme dimerization and the nucleotide-protein contact mediated by Tyr-5 side chain residue helps the dimerization process. Analysis of ATPase activities of mutant proteins provides evidence that dimerization enhances the ATP-hydrolysis turnover. The structure of the Y5S mutant of the N-terminal 43-kDa fragment of E. coli DNA GyrB subunit indicates that Tyr-5 residue provides a scaffold for the ATP-hydrolysis center. We describe a channel formed at the dimer interface that provides a structural mechanism to allow reactive water molecules to access the gamma-phosphate group of the bound ATP molecule. Together, these results demonstrate that dimerization strongly contributes to the folding and stability of the catalytic site for ATP hydrolysis. A role for the essential Mg(2+) ion for the orientation of the phosphate groups of the bound nucleotide inside the reactive pocket was also uncovered by superposition of the 5'-adenylyl beta-gamma-imidodiphosphate (ADPNP) wild-type structure to the salt-free ADPNP structure.

Adenosine Triphosphatases↗

PubMed1997

A 'specificity' pocket inferred from the crystal structures of the complexes of aldose reductase with the pharmaceutically important inhibitors tolrestat and sorbinil.

BACKGROUND: Aldose reductase (AR) is an NADPH-dependent enzyme implicated in long-term diabetic complications. Buried at the bottom of a deep hydrophobic cleft, the NADPH coenzyme is surrounded by the conserved hydrophilic residues of the AR active site. The existence of an anionic binding site near the NADP+ has been determined from the structures of the complexes of AR with citrate, cacodylate and glucose-6-phosphate. The inhibitor zopolrestat binds to this anionic site, and in the hydrophobic cleft, after a change of conformation which opens a 'specificity' pocket. RESULTS: The crystal structures of the porcine AR holoenzyme and its complexes with the inhibitors tolrestat and sorbinil have been solved; these structures are important as tolrestat and sorbinil are, pharmaceutically, the most well-studied AR inhibitors. The active site of the holoenzyme was analyzed, and binding of the inhibitors was found to involve two contact zones in the active site: first, a recognition region for hydrogen-bond acceptors near the coenzyme, with three centers, including the anionic site; and second, a hydrophobic contact zone in the active-site cleft, which in the case of tolrestat includes the specificity pocket. The conformational change leading to the opening of the specificity pocket upon tolrestat binding is different to the one seen upon zopolrestat binding; this pocket binds inhibitors that are more effective against AR than against aldehyde reductase. CONCLUSIONS: The active site of AR adapts itself to bind tightly to different inhibitors; this happens both upon binding to the inhibitor's hydrophilic heads, and at the hydrophobic and specificity pockets of AR, which can change their shape through different conformational changes of the same residues. This flexibility could explain the large variety of possible substrates of AR.

Aldehyde Reductase↗

PubMed1997

A multicopy modeling of the water distribution in macromolecular crystals.

A multicopy protocol is proposed for modeling macromolecular hydration using diffraction experimental data (X-ray or neutron) to search for a better description of delocalized water sites than that given by point water models. The model consists of one macro-molecule and several copies of each water molecule, refined simultaneously against diffraction data using molecular dynamics techniques. The protocol was applied to BPTI and an RNA tetradecamer. The sites defined by the different copies range from very ordered ones to continuous channels; they fit the density maps and agree with the diffraction amplitudes with an accuracy comparable with usual crystallographic methods. The delocalization of water in channels agrees with the high mobility observed in NMR experiments.

Aprotinin↗

PubMed1995

On the solution of the molecular-replacement problem at very low resolution: application to large complexes.

The applicability of the molecular-replacement (MR) method, implemented through the AMoRe package [Navaza (1994). Acta Cryst. A50, 157-163], is studied at very low resolution (d > 20 A) and for very large molecular complexes. Due to the nature of the low-resolution data, specific problems appear. In particular, rotation-function peaks are very broad and translation functions based on Patterson overlap show large spurious peaks. To solve these problems, the translation function is replaced by a search using amplitude correlation and a systematic three-dimensional angular search is performed around each rotation-function peak. Furthermore, these functions are applied in different resolution ranges during the same search. The corresponding algorithms are applied to two cases: the tRNA(Asp)-synthetase complex (neutron diffraction data) and a ribosome model crystal (calculated data). This new implementation is shown to solve the problem for a variety of search models, ranging from a detailed atomic model to a rough envelope.

Journal Article↗

PubMed1991

Crystal structure of calf eye lens gamma-crystallin IIIb at 2.5 A resolution: its relation to function.

The crystal structure of gamma-crystallin IIIb (gamma C) from calf eye lens has been refined at 2.5 A resolution. The molecule of about 21 kDa consists of two similar domains. Each domain is composed of two motifs with the 'Greek key' topology which form a pair of four-stranded beta-sheets with an antiparallel packing. The molecule has three hydrophobic cores: one within each domain and one between them. Six of the eight functionally important cysteines are located within the N-domain, and only two in the C-domain. Several large clusters of charged residues are at the surface of the molecule. Surface residues Val 101, Met 103 and Leu 155 are important for packing of molecules in crystal medium and possibly in the lens. Features of the gamma-crystallin IIIb molecule which may be related to its function in the vertebrate eye lens are briefly discussed. An attempt has been made to correlate molecular characteristics with some general properties of the eye lens such as high density and refractive index gradients and strong stability of the lens during an organism's lifetime.

Amino Acid Sequence↗