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PubMed1995

Mechanically stretched chromosomes as targets for high-resolution FISH mapping.

When used with metaphase chromosomes, fluorescence in situ hybridization (FISH) makes it possible to localize probes to individual chromosome bands and to establish the order of probes separated by > or = 2-3 Mb in dual-color hybridizations. We evaluated the use of mechanically stretched chromosomes as hybridization targets for increased mapping resolution. Mapping resolution was tested by pair-wise hybridizations with probes from the 1p32-p33 region, spanning distances from 20 to approximately 1500 kb. Probes separated by > or = 170 kb could be ordered relative to one another and to the centromere-telomere axis of the chromosome. The advantages of the technique are the simple procedure for preparing the slides, the straightforward interpretation of the results, and the ability to score the predominant order from < 10 stretched chromosomes. However, because of the variability of stretching from one sample to another, the calculation of actual physical distances between probes is not possible. To illustrate the utility of this method, we showed that the gene for receptor tyrosine kinase TIE lies centromeric to COL9A2, RLF, and L-MYC genes at 1p32. The use of mechanically stretched chromosomes provides < or = 10-fold increased mapping resolution as compared with conventional metaphase FISH. Thus, the technique effectively bridges the gap between metaphase mapping and ultra-high-resolution mapping (1-300 kb) techniques, such as the DNA fiber FISH.

Adult↗

PubMed1998

A high-resolution microsatellite map of the mouse genome.

The European Collaborative Interspecific Backcross (EUCIB) resource was constructed for the purposes of high-resolution genetic mapping of the mouse genome (). The large Mus spretus/C57BL/6 backcross of 982 progeny has a genetic resolution of 0.3 cM at the 95% confidence level ( approximately 500 kb in the mouse genome). We have used the EUCIB mapping resource to develop a genome-wide high-resolution genetic map incorporating 3368 microsatellites. The microsatellites are distributed among 2302 genetically separated bins with 1.46 markers per bin on average. Average bin separation is 0.61 cM. This high-resolution genetic map will aid the construction of a robust physical map of the mouse genome.

Animals↗

PubMed2003

Analytic explanation of spatial resolution related to bandwidth and detector aperture size in thermoacoustic or photoacoustic reconstruction.

An analytic explanation of the spatial resolution in thermoacoustic or photoacoustic reconstruction is presented. Three types of specific recording geometries, including spherical, planar, and cylindrical surface, as well as other general cases, are investigated. Analytic expressions of the point-spread functions (PSF's), as a function of the bandwidth of the measurement system and the finite size of the detector aperture, are derived based on rigorous reconstruction formulas. The analyses clearly reveal that the dependence of the PSF's on the bandwidth of all recording geometries shares the same space-invariant expression while the dependence on the aperture size of the detector differs. The bandwidth affects both axial and lateral resolutions; in contrast, the detector aperture blurs the lateral resolution greatly but the axial resolution only slightly.

Acoustics↗

PubMed2003

Numerical viscosity and resolution of high-order weighted essentially nonoscillatory schemes for compressible flows with high Reynolds numbers.

A quantitative study is carried out in this paper to investigate the size of numerical viscosities and the resolution power of high-order weighted essentially nonoscillatory (WENO) schemes for solving one- and two-dimensional Navier-Stokes equations for compressible gas dynamics with high Reynolds numbers. A one-dimensional shock tube problem, a one-dimensional example with parameters motivated by supernova and laser experiments, and a two-dimensional Rayleigh-Taylor instability problem are used as numerical test problems. For the two-dimensional Rayleigh-Taylor instability problem, or similar problems with small-scale structures, the details of the small structures are determined by the physical viscosity (therefore, the Reynolds number) in the Navier-Stokes equations. Thus, to obtain faithful resolution to these small-scale structures, the numerical viscosity inherent in the scheme must be small enough so that the physical viscosity dominates. A careful mesh refinement study is performed to capture the threshold mesh for full resolution, for specific Reynolds numbers, when WENO schemes of different orders of accuracy are used. It is demonstrated that high-order WENO schemes are more CPU time efficient to reach the same resolution, both for the one-dimensional and two-dimensional test problems.

Journal Article↗

PubMed2002

Atomic resolution three-dimensional electron diffraction microscopy.

We report the development of a novel form of diffraction-based 3D microscopy to overcome resolution barriers inherent in high-resolution electron microscopy and tomography. By combining coherent electron diffraction with the oversampling phasing method, we show that the 3D structure of a nanocrystal can be determined ab initio at a resolution of 1 A from 29 simulated noisy diffraction patterns. This new form of microscopy can be used to image the 3D structures of nanocrystals and noncrystalline samples, with resolution limited only by the quality of sample diffraction.

Journal Article↗

PubMed2001

Sparsity of the normal matrix in the refinement of macromolecules at atomic and subatomic resolution.

The normal matrix in the least-squares refinement of macromolecules is very sparse when the resolution reaches atomic and subatomic levels. The elements of the normal matrix, related to coordinates, thermal motion and charge-density parameters, have a global tendency to decrease rapidly with the interatomic distance between the atoms concerned. For instance, in the case of the protein crambin at 0.54 A resolution, the elements are reduced by two orders of magnitude for distances above 1.5 A. The neglect a priori of most of the normal-matrix elements according to a distance criterion represents an approximation in the refinement of macromolecules, which is particularly valid at very high resolution. The analytical expressions of the normal-matrix elements, which have been derived for the coordinates and the thermal parameters, show that the degree of matrix sparsity increases with the diffraction resolution and the size of the asymmetric unit.

Aldehyde Reductase↗

PubMed1995

Use of globic scattering factors for protein structures at low resolution.

At 3 to 4 A resolution, the electron density of a protein may be modeled by a continuous chain of 'globs' representing the amide region of the peptide backbone and the side-chain residues. Group scattering factors are derived from a trans planar C alpha C = ONC alpha backbone segment and most favored side-chain conformer for 18 different amino acids. Trial calculations indicate that the phase error and crystallographic residual comparing the atomic and 'globic' models rapidly decrease from high to low resolution. At 3 A resolution, the phase error is approximately 80 degrees. These results indicate that the electron density of a protein composed of N amino acid residues may be adequately modeled by 2N globs at low resolution.

Amino Acids↗

PubMed1992

Structure determination and refinement of homotetrameric hemoglobin from Urechis caupo at 2.5 A resolution.

A 5 A resolution multiple isomorphous replacement solution for hemoglobin isolated from Urechis caupo revealed a previously unobserved quaternary structure for tetrameric hemoglobin [Kolatkar, Meador, Stanfield & Hackert (1988). J. Biol. Chem. 263(7), 3462-3465]. We report here the structure of Urechis hemoglobin in the cyanomet state refined to 2.5 A resolution by simulated annealing yielding R = 0.148 for reflections F greater than 3 sigma between 5.0 and 2.5 A resolution. The starting model was fitted to a map originally derived from multiple-wavelength anomalous-dispersion phases to 3 A resolution that was then subjected to cyclic twofold molecular averaging and solvent flattening. Structural analysis of the resultant model shows that the unique quaternary assemblage is possible due to several favorable interactions between subunits, including salt links, hydrophobic pockets and interactions mediated by bound water. The tetramer is stabilized by subunit-subunit interactions between the G/H turns and D helices within the crystallographic dimer, and the A/B turn regions and E helices between subunits related by a molecular twofold axis. Interestingly, each subunit has one cysteine residue (Cys21) located in the A/B turn. These twofold-related cysteinyl residues are near enough to one another to form a disulfide bridge but do not.

Animals↗

PubMed2000

Low-resolution phase extension using wavelet analysis.

A method to extend low-resolution phases is presented which uses histogram matching not only of the electron density, but also of histograms obtained from the different levels of detail provided by the wavelet transform of the electron density. Statistical values for the wavelet coefficients can be predicted and depend only on the resolution and solvent content. Therefore, new details can be added to an electron-density map by matching the values of the wavelet coefficients to those predicted for an increased resolution. The positions of the new details are also guided by the diffraction pattern. In this way, the resolution can be increased gradually; on a number of trial structures of different size, solvent percentage and space group, it has been possible to extend the phasing from 10 A to around 6-7 A.

Electrons↗

PubMed2001

X-ray structure of bovine pancreatic phospholipase A2 at atomic resolution.

Using synchrotron radiation and a CCD camera, X-ray data have been collected from wild-type bovine pancreatic phospholipase A(2) at 100 K to 0.97 A resolution allowing full anisotropic refinement. The final model has a conventional R factor of 9.44% for all reflections, with a mean standard uncertainty for the positional parameters of 0.031 A as calculated from inversion of the full positional least-squares matrix. At 0.97 A resolution, bovine pancreatic phospholipase A(2) reveals for the first time that its rigid scaffolding does not preclude flexibility, which probably plays an important role in the catalytic process. Functionally important regions (the interfacial binding site and calcium-binding loop) are located at the molecular surface, where conformational variability is more pronounced. A cluster of 2-methyl-2,4-pentanediol molecules is present at the entrance of the hydrophobic channel that leads to the catalytic site and mimics the fatty-acid chains of a substrate analogue. Bovine pancreatic phospholipase A(2) at atomic resolution is compared with previous crystallographic structures and with models derived from nuclear magnetic resonance studies. Given the high structural similarity among extracellular phospholipases A(2) observed so far at lower resolution, the results arising from this structural analysis are expected to be of general validity for this class of enzymes.

Animals↗

PubMed2001

X-ray structure of nitric oxide reductase (cytochrome P450nor) at atomic resolution.

Crystal structures of the nitric oxide reductase cytochrome P450nor (P450nor) in the ferric resting and the ferrous carbonmonoxy (CO) states have been determined at 1.00 and 1.05 A resolution, respectively. P450nor consists of 403 amino-acid residues (46 kDa) and is one of the largest proteins refined to this resolution so far. The final models have conventional R factors of 10.2% (ferric resting) and 11.7% (ferrous CO), with mean coordinate errors of 0.028 (ferric resting) and 0.030 A (ferrous CO) as calculated from inversion of the full positional least-squares matrix. Owing to the atomic resolution, novel features are found in the refined structures. Firstly, two orientations of the haem are observed both in the ferric resting and the ferrous CO states. Secondly, a disordered water molecule bound to the haem iron is found in the ferric resting state. In addition, the accurate structures at atomic resolution enabled the examination of general stereochemical parameters that are commonly used in refinement cycles of protein structures.

Amino Acids↗

PubMed2002

Alternate conformations observed in catalytic serine of Bacillus subtilis lipase determined at 1.3 A resolution.

Bacillus subtilis extracellular lipase (BsL) has an exceptionally low molecular weight (19.4 kDa) for a member of the lipase family. A crystallographic study was performed on BsL in order to design and produce mutant BsL that will be more suitable for industrial uses based on analysis of the three-dimensional structure. Recently, the crystal structure of BsL has been determined at 1.5 A resolution [van Pouderoyen et al. (2001). J. Mol. Biol. 309, 215-226]. In the present study, a new crystal form of BsL which provides diffraction data to higher resolution was obtained and its structure was determined at 1.3 A using the MAD method. It was found that the active-site residue Ser77 has alternate side-chain conformations. The O(gamma) atom of the first conformer forms a hydrogen bond to the N(epsilon) atom of His155, a member of the catalytic triad. In contrast, the second conformer is constructed with a hydrogen bond to the side-chain atom of the adjacent His76. These two conformers presumably correspond to the active and inactive states, respectively. Similar alternate conformations in the catalytic serine residue have been observed in Fusarium solani cutinase determined at 1.0 A resolution and Penicillium purpurogenum acetylxylan esterase at 0.9 A resolution. In addition, a glycerol molecule, which was used as a cryoprotectant, is found to be located in the active site. On the basis of these results, a model for substrate binding in the reaction-intermediate state of BsL is proposed.

Amino Acids↗

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↗

PubMed2003

Direct experimental observation of the hydrogen-bonding network of a glycosidase along its reaction coordinate revealed by atomic resolution analyses of endoglucanase Cel5A.

Non-covalent interactions between protein and ligand at the active centre of glycosidases play an enormous role in catalysis. Dissection of these hydrogen-bonding networks is not merely important for an understanding of enzymatic catalysis, but is also increasingly relevant for the design of transition-state mimics, whose tautomeric state, hydrogen-bonding interactions and protonation contribute to tight binding. Here, atomic resolution ( approximately 1 A) analysis of a series of complexes of the 34 kDa catalytic core domain of the Bacillus agaradhaerens endoglucanase Cel5A is presented. Cel5A is a 'retaining' endoglucanase which performs catalysis via the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. Previous medium-resolution analyses of a series of enzymatic snapshots has revealed conformational changes in the substrate along the reaction coordinate (Davies et al., 1998). Here, atomic resolution analyses of the series of complexes along the pathway are presented, including the 'Michaelis' complex of the unhydrolysed substrate, the covalent glycosyl-enzyme intermediate and the complex with the reaction product, cellotriose. These structures reveal intimate details of the protein-ligand interactions, including most of the carbohydrate-associated H atoms and the tautomeric state of crucial active-centre groups in the pH 5 orthorhombic crystal form and serve to illustrate the potential for atomic resolution analyses to inform strategies for enzyme inhibition.

Bacillus↗

PubMed2003

Improving macromolecular atomic models at moderate resolution by automated iterative model building, statistical density modification and refinement.

An iterative process for improving the completeness and quality of atomic models automatically built at moderate resolution (up to about 2.8 A) is described. The process consists of cycles of model building interspersed with cycles of refinement and combining phase information from the model with experimental phase information (if any) using statistical density modification. The process can lead to substantial improvements in both the accuracy and completeness of the model compared with a single cycle of model building. For eight test cases solved by MAD or SAD at resolutions ranging from 2.0 to 2.8 A, the fraction of models built and assigned to sequence was 46-91% (mean of 65%) after the first cycle of building and refinement, and 78-95% (mean of 87%) after 20 cycles. In an additional test case, an incorrect model of gene 5 protein (PDB code 2gn5; r.m.s.d. of main-chain atoms from the more recent refined structure 1vqb at 1.56 A) was rebuilt using only structure-factor amplitude information at varying resolutions from 2.0 to 3.0 A. Rebuilding was effective at resolutions up to about 2.5 A. The resulting models had 60-80% of the residues built and an r.m.s.d. of main-chain atoms from the refined structure of 0.20 to 0.62 A. The algorithm is useful for building preliminary models of macromolecules suitable for an experienced crystallographer to extend, correct and fully refine.

Algorithms↗

PubMed2003

Statistical analysis of crystallographic data obtained from squid ganglion DFPase at 0.85 A resolution.

The X-ray crystal structure of squid-type diisopropylfluorophosphatase (DFPase) has been refined to a resolution of 0.85 A and a crystallographic R value of 9.4%. Crystal annealing improved both the mosaicity and resolution of the crystals considerably. The overall structure of this protein represents a six-bladed beta-propeller with two calcium ions bound in a central water-filled tunnel. 496 water, two glycerol and two MES buffer molecules and 18 PEG fragments of different lengths could be refined in the solvent region. 45 of the 314 residues have been refined with alternative orientations. H atoms have been omitted from disordered residues. For the residues of the inner beta-strands, H atoms are visible in a normal F(o) - F(c) difference map of a hydrogen-deficient structure model. The 208 most reliable residues, without disorder or reduced occupancy in their side chains, were finally refined without restraints. A subsequent full-matrix refinement cycle for the positional parameters yielded estimated standard deviations (e.s.d.s) by matrix inversion. The thus calculated bond lengths and bond angles and their e.s.d.s were used to obtain averaged bond lengths and bond angles, which were compared with the restraints applied in the preceding refinement cycles. The lengths and angles of the hydrogen bonds inside the antiparallel beta-sheets of the DFPase structure were compared with data averaged over 11 high-resolution protein structures. Torsion angles were averaged according to angle types used as restraints in X-PLOR and CNS and subsequently compared with values obtained from 46 high-resolution structures. Side-chain torsion angles were also classified into rotamer types according to the Penultimate Rotamer Library. Moreover, precise dimensions for both Ca(2+)-coordination polyhedra could be obtained and the coordination of one Ca(2+) ion by an imidazole N atom was confirmed. This statistical analysis thus provides a first step towards a set of restraints that are founded completely on macromolecular data; however, 10-20 additional protein data sets of comparable accuracy and size will be required to obtain a larger statistical base, especially for side-chain analysis.

Amino Acids↗

PubMed2003

Direct phasing at low resolution of a protein copurified with human paraoxonase (PON1).

In this paper, the low-resolution structure of a previously unknown protein copurified with human paraoxonase (PON1) is reported. The structure of this protein was very difficult to solve using classical crystallographic methods. Progress was made using a new phasing method based on topological analysis. From the experimental point of view, this method has the advantage of requiring only a simple low-resolution X-ray data set. The program used and the different steps of the data-processing and phasing procedure are described. The results provided an insight into the failure of previous molecular-replacement attempts. The low-resolution shape of the protein which was presented with confidence is compared with and confirmed by the structure at 1.8 A solved subsequently using classical methods. This work shows that this direct-phasing method could be used systematically in difficult cases: it provides low-resolution structural information comparable with that obtainable by electron microscopy.

Aryldialkylphosphatase↗

PubMed2005

The crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase from Escherichia coli with improved resolution show new features of biological significance.

Dihydrodipicolinate synthase (DHDPS) mediates the key first reaction common to the biosynthesis of (S)-lysine and meso-diaminopimelate. The activity of DHDPS is allosterically regulated by the feedback inhibitor (S)-lysine. The crystal structure of DHDPS from Escherichia coli has previously been published, but to only a resolution of 2.5 A, and the structure of the lysine-bound adduct was known to only 2.94 A resolution. Here, the crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase from E. coli are presented to 1.9 and 2.0 A, respectively, resolutions that allow, in particular, more accurate definition of the protein structure. The general architecture of the active site is found to be consistent with previously determined structures, but with some important differences. Arg138, which is situated at the entrance of the active site and is thought to be involved in substrate binding, has an altered conformation and is connected via a water molecule to Tyr133 of the active-site catalytic triad. This suggests a hitherto unknown function for Arg138 in the DHDPS mechanism. Additionally, a re-evaluation of the dimer-dimer interface reveals a more extensive network of interactions than first thought. Of particular interest is the higher resolution structure of DHDPS with (S)-lysine bound at the allosteric site, which is remote to the active site, although connected to it by a chain of conserved water molecules. (S)-Lysine has a slightly altered conformation from that originally determined and does not appear to alter the DHDPS structure as others have reported.

Allosteric Site↗