Biomedical subjects

Miroslawa Dauter

Publications and source records attributed to Miroslawa Dauter.

7 recordsLinked to original sources

PubMed2004

Crystal structure of the YffB protein from Pseudomonas aeruginosa suggests a glutathione-dependent thiol reductase function.

BACKGROUND: The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. RESULTS: The structure was determined at 1.0 A resolution by single-wavelength anomalous diffraction. The fold is very similar to that of arsenate reductase, which is an extension of the thioredoxin fold. CONCLUSION: Given the conservation of the functionally important residues and the ability to bind glutathione, YffB is likely to function as a GSH-dependent thiol reductase.

Arsenite Transporting ATPases↗

PubMed2003

Phasing on anomalous signal of sulfurs: what is the limit?

Recent years have witnessed significant advancements in X-ray data-acquisition techniques and phasing algorithms, which have made possible the successful use of a very small anomalous diffraction signal for the solution of crystal structures of macromolecules. Two crystal structures, a 44 kDa glucose isomerase containing nine sulfurs and a 33 kDa xylanase containing five sulfurs, have been solved from single-wavelength anomalous data using widely available methods and programs. These two enzymes contain less sulfur than most proteins in the bacterial or eukaryotic proteomes, providing a Bijvoet ratio of about 0.6%. For glucose isomerase the automatically interpretable electron-density maps could be obtained at high as well as low resolution. The S-SAD approach relies on the anomalous signal of sulfur naturally occurring in proteins and alleviates all need for sample derivatization. It may therefore be applicable to all protein crystals able to provide accurate diffraction data.

Aldose-Ketose Isomerases↗

PubMed2003

SAD manganese in two crystal forms of glucose isomerase.

Glucose isomerase from Streptomyces rubiginosus was crystallized in two forms: I222, with one molecule of 44 kDa in the asymmetric unit, and P2(1)2(1)2, with two unique molecules. The I222 structure is known, but the P2(1)2(1)2 form has not been solved before. X-ray diffraction data for the P2(1)2(1)2 form were collected at a wavelength of 1.54 A and data for the I222 form were collected at three different wavelengths: 1.34, 1.07 and 0.98 A. The amount of anomalous signal from one Mn and eight S atoms in these data sets varies from 1.24% to as low as 0.56%. The dual-space direct-methods program SHELXD, run against the Bijvoet differences, gave a clear solution of all anomalous scatterers for all data sets. The Mn positions only were used for SAD phasing of all four data sets. The electron-density map after density modification, resulting from the phasing of a single-wavelength data set and based purely on the anomalous deltaf" contribution, was clearly interpretable; an almost complete model of the protein was built by wARP without human intervention in all four cases. As far as is known, this is the first time that an anomalous signal as low as 0.6% has successfully been used to determine the structure of a macromolecule.

Aldose-Ketose Isomerases↗

PubMed2003

Phasing at high resolution using Ta6Br12 cluster.

The Ta(6)Br(12)(2+) cluster compound is known to be a powerful reagent for derivatization of crystals of large macromolecules at low resolution. The cluster is a regular octahedron of six Ta atoms with 12 bridging Br atoms at the edges of the octahedron. The cluster is compact, of approximately spherical shape, with a radius of about 6 A. Both tantalum and bromine display a significant anomalous diffraction signal at their absorption edges at 1.25 and 0.92 A, respectively. At resolutions lower than 5 A the tantalum cluster behaves as a super-atom and provides very large isomorphous and anomalous signals, which significantly diminish at about 4 A. However, beyond 3 A the individual Ta atoms can be resolved and the phasing power of the cluster increases again. The Ta(6)Br(12)(2+) cluster has been used for phasing four different proteins at high resolution. Ta(6)Br(12)(2+) appeared to be a mild derivatization reagent and, despite partial incorporation, led to a successful solution of crystal structures by the single-wavelength anomalous diffraction (SAD) approach.

Bromides↗

PubMed2002

Locating the anomalous scatterer substructures in halide and sulfur phasing.

Improved data quality now makes it feasible to exploit the weak anomalous signal derived only from the sulfurs inherent to the protein or in particular from halide ions incorporated by soaking. The latter technique requires the location of a high number of partially occupied halide sites. This number appears to be roughly proportional to the exposed protein surface. This paper explores the application of dual-space ab initio methods as implemented in the program SHELXD to the location of substructures of sulfur in SAD experiments, bromide in SAD and MAD experiments and iodide using SAD and SIRAS to determine the anomalous-atom substructure. Sets of atoms consistent with the Patterson function were generated as a starting point for the dual-space recycling procedure in SHELXD. The substructure is then expanded to the full structure by maximum-likelihood phasing with SHARP and density modification with the program DM. Success in the location of the substructures and subsequent phasing depends critically on the quality of the data and on the extent of the anomalous signal. This varies with each crystal and soak, but for the same crystal the significance of the anomalous signal was found to be highly sensitive to the redundancy of the intensity measurements, which in some cases made all the difference. This is illustrated by the determination of the previously unknown structure of repeat 11 of the human mannose-6-phosphate/insulin-like growth factor II receptor (Man6P/IGFII-receptor), with 310 amino acids in the asymmetric unit, which was phased by soaking the crystals in a cryoprotectant solution containing halide anions.

Amino Acid Sequence↗

PubMed2002

Atomic resolution structure of Erwinia chrysanthemi L-asparaginase.

An X-ray structure of L-asparaginase from Erwinia chrysanthemi (ErA) has been refined at 1 A resolution to an R factor of below 0.1, using data collected on a synchrotron source. With four molecules of the enzyme consisting of 327 amino acids each, this crystal contains one of the largest asymmetric units of a protein refined to date at atomic resolution. Previously, structures of ErA and of related enzymes from other bacterial sources have been refined at resolutions not exceeding 1.7 A; thus, the present structure represents a very significant improvement in the quality of the available models of these proteins and should provide a good basis for future studies of the conformational variability of proteins, identification of subtle conformational features and corroboration of the stereochemical libraries, amongst other things. L-Asparaginases, which are enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 y as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia, although the details of the enzymatic reaction and substrate specificity have not yet been completely elucidated. This atomic resolution structure is a step in that direction.

Asparaginase↗

PubMed2002

Jolly SAD.

Examples of phasing macromolecular crystal structures based on single-wavelength anomalous dispersion (SAD) show that this approach is more powerful and may have more general application in structural biology than was anticipated. Better data-collection facilities and cryogenic techniques, coupled with powerful programs for data processing, phasing, density modification and automatic model building, means that the SAD approach may gain wide popularity owing to its simplicity, less stringent wavelength requirements and faster data collection and phasing than the multi-wavelength (MAD) approach. It can be performed at any wavelength where anomalous scattering can be observed, in many cases using laboratory X-ray sources.

Aldose-Ketose Isomerases↗