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At least 973 records · Page 54Linked to original sources
PubMed1998

Crystallization and preliminary X-ray analysis of recombinant glutamate mutase and of the isolated component S from Clostridium cochlearium.

Glutamate mutase [varepsilon2sigma2(B12)1] was reconstituted by incubating purified components E (varepsilon2) and S (sigma2) from Clostridium cochlearium, both produced in Escherichia coli, with either aquo- or cyanocobalamin. The inactive glutamate mutase obtained was crystallized with polyethyleneglycol 4000 as precipitant. Crystals are monoclinic with space group P21 and have cell dimensions a = 64.6, b = 113.2, c = 108.4 A and beta = 96.0 degrees for the glutamate mutase reconstituted with aquocobalamin. They diffract to a resolution of at least 2.7 A. Isolated component S was crystallized in the presence of an excess of cyanocobalamin, yielding red crystals of space group I422 with unit-cell dimensions of a = b = 69.9 and c = 107.1 A. The crystals diffract to about 3.2 A resolution. Native data sets were collected for both crystal forms.

Bacterial Proteins↗
PubMed1998

Structure of glucoamylase from Saccharomycopsis fibuligera at 1.7 A resolution.

The yeast Saccharomycopsis fibuligera produces a glucoamylase which belongs to sequence family 15 of glycosyl hydrolases. The structure of the non-glycosyl-ated recombinant enzyme has been determined by molecular replacement and refined against 1.7 A resolution synchrotron data to an R factor of 14.6%. This is the first report of the three-dimensional structure of a yeast family 15 glucoamylase. The refinement from the initial molecular-replacement model was not straightforward. It involved the use of an unrestrained automated refinement procedure (uARP) in combination with the maximum-likelihood refinement program REFMAC. The enzyme consists of 492 amino-acid residues and has 14 alpha-helices, 12 of which form an (alpha/alpha)6 barrel. It contains a single catalytic domain but no starch-binding domain. The fold of the molecule and the active site are compared to the known structure of the catalytic domain of a fungal family 15 glucoamylase and are shown to be closely similar. The active- and specificity-site residues are especially highly conserved. The model of the acarbose inhibitor from the analysis of the fungal enzyme fits tightly into the present structure. The active-site topology is a pocket and hydrolysis proceeds with inversion of the configuration at the anomeric carbon. The enzyme acts as an exo-glycosyl hydrolase. There is a Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] molecule acting as an inhibitor in the active-site pocket.

Amino Acid Sequence↗
PubMed1998

Crystallization and preliminary X-ray analysis of the beta-isoform of glutamate decarboxylase from Escherichia coli.

Glutamate decarboxylase (GAD) is a vitamin B6 enzyme which catalyzes the alpha-decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Escherichia coli cells coexpress two highly homologous enzyme isoforms, GADalpha and GADbeta. Well diffracting crystals of GADbeta were obtained by taking advantage of the possibility of expressing each isoform separately. They belong to space group P31 or P32 with the unit-cell dimensions a = b = 115.6 and c = 206.6 A and contain one GAD hexamer in the asymmetric unit. High-resolution synchrotron data were collected at 100 K for the native protein and a potential heavy-atom derivative.

Bacterial Proteins↗
PubMed1999

Cloning, overexpression, crystallization and preliminary X-ray analysis of a family 1 beta--glucosidase from Streptomyces.

An intracellular beta-glucosidase (Bgl3) from Streptomyces sp. has been cloned and overexpressed in Escherichia coli. The introduction of a His tag at the N-terminal end of the protein has allowed its purification to homogeneity by a single chromatographic step, with yields of 150-200 mg of pure protein per litre of E. coli culture. The enzyme (52.6 kDa) is a retaining glycosidase able to hydrolyze a wide range of disaccharides and oligosaccharides and to perform transglycosylation. Crystals of recombinant Bgl3 have been grown from an ammonium sulfate solution using the hanging-drop vapour-diffusion method at 293 K. The crystals belong to the orthorhombic space group I222 with unit-cell dimensions a = 101.6, b = 113.4 and c = 187.5 A at room temperature and contain two molecules per asymmetric unit. A full 1.69 A resolution diffraction data set (97.7% completeness) has been collected from frozen crystals in a solution containing 30% sucrose, using synchrotron radiation.

Base Sequence↗
PubMed1999

Meaningful refinement of polyalanine models using rigid-body simulated annealing: application to the structure determination of the A31P Rop mutant.

Conventional refinement methods, when applied to even correctly positioned polyalanine models of a target structure, result in a systematic distortion of the molecular geometry and to a concomitant increase in the mean phase difference from the correct phase set. Here, it is shown that iterative rigid-body simulated-annealing refinement of polyalanine models employing successively fewer residues per rigid body (down to one alanine residue per body) at a very high initial temperature (of the order of T0 = 10000 K) and with the geometric energy terms switched on, not only preserves the geometry of the model but can also converge to an essentially correct polyalanine trace of the target structure, even when the starting model deviates systematically and significantly from the sought structure. As an example of the application of the method, details are presented of the structure determination of the Ala31Pro mutant of the Rop protein, where an initial roughly positioned polyalanine model (giving an average phase difference of 78.2 degrees from the final phase set) was successfully refined against a 1.8 A resolution native data set, leading to an essentially correct model of the main chain with an average displacement of its atomic positions from the final model of 0.275 A. The phases calculated from this refined polyalanine model had an average difference of 43.8 degrees from the final phase set (corresponding to a mean figure of merit of 0.72) and gave a readily interpretable electron-density map.

Bacterial Proteins↗
PubMed1999

Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain.

The C-terminal two-thirds segment of integrase derived from the simian immunodeficiency virus has been cloned, expressed in Escherichia coli, and purified to greater than 95% homogeneity. The protein encompasses amino-acid residues 50-293 and contains a F185H substitution to enhance solubility. In dilute solutions at concentrations below 1 mg ml(-1), the enzyme is predominantly dimeric. At the higher concentrations (>10 mg ml(-1)) required to enable crystallization, the enzyme self-associates to form species with molecular weights greater than 200 kDa. Despite the apparent high aggregation in solution, the enzyme crystallizes from a 8%(v/v) polyethylene glycol (molecular weight 6000) solution in a form suitable for X-ray diffraction studies. The resulting single crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 79.76, b = 99.98, c = 150.2 A, alpha = beta = gamma = 90 degrees and Z = 4. Under X-ray irradiation generated with a rotating-anode generator, the crystals diffract to 2.8 A resolution and allow collection of a native 3 A resolution diffraction data set.

Cloning, Molecular↗
PubMed1999

Cryocrystallography of a Kunitz-type serine protease inhibitor: the 90 K structure of winged bean chymotrypsin inhibitor (WCI) at 2.13 A resolution.

The crystal structure of a Kunitz-type double-headed alpha--chymotrypsin inhibitor from winged bean seeds has been refined at 2.13 A resolution using data collected from cryo-cooled (90 K) crystals which belong to the hexagonal space group P6(1)22 with unit-cell parameters a = b = 60.84, c = 207.91 A. The volume of the unit cell is reduced by 5.3% on cooling. The refinement converged to an R value of 20.0% (R(free) = 25.8%) for 11100 unique reflections and the model shows good stereochemistry, with r.m.s. deviations from ideal values for bond lengths and bond angles of 0.011 A and 1.4 degrees, respectively. The structural architecture of the protein consists of 12 antiparallel beta-strands joined in the form of a characteristic beta-trefoil fold, with the two reactive-site regions, Asn38-Leu43 and Gln63-Phe68, situated on two external loops. Although the overall protein fold is the same as that of the room-temperature model, some conformational changes are observed in the loop regions and in the side chains of a few surface residues. A total of 176 ordered water molecules and five sulfate ions are included in the model.

Binding Sites↗
PubMed2000

Crystallization and preliminary X-ray analysis of an intracellular xylanase from Bacillus stearothermophilus T-6.

Xylanases (1,4-beta-D-xylan xylanhydrolases; E.C. 3.2.1.8) hydrolyze the 1,4-beta-D-xylopyranosyl linkage of xylans. The structural characterization of xylanase active sites is of great interest, since it can lead to a better understanding of their catalytic mechanism and contribute significant knowledge to the rational design of specific oligosaccharide-binding sites via protein engineering. An intracellular xylanase gene (xynA2) from Bacillus stearothermophilus T-6 has recently been cloned and sequenced. The xynA2 gene encodes for an intracellular enzyme (IXT6) of 331 amino acids, with a calculated molecular weight of 38 639 Da and a pI of 5.72. Based on sequence homology, the enzyme belongs to family 10 of the glycosyl hydrolases. The xynA2 gene product (IXT6) was overproduced in Escherichia coli and purified to homogeneity. Crystallographic studies of IXT6 were initiated in order to study the specificity and mechanism of catalysis of this unique xylanase, as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. The M1 crystal form was found to be the most suitable for detailed crystal structure analysis. These crystals belong to a C--centered monoclinic crystal system (space group C2) with unit-cell parameters a = 170.6, b = 82.5, c = 80.0 A, beta = 91.43 degrees. They are mechanically strong, are fairly stable in the X-ray beam and diffract X--rays to better than 2.5 A resolution. A full 2.9 A resolution diffraction data set (97.9% completeness, R(merge) = 8.4%) has recently been collected from one crystal at room temperature using X-ray synchrotron radiation (lambda = 1.125 A) and a MAR300 imaging-plate area detector. A comparable 2.5 A data set was measured at 90 K using a rotating-anode X-ray source and an R-AXIS IIc imaging-plate area detector (97.2% completeness, R(merge) = 6.9%). Molecular-replacement studies and multiple anomalous dispersion (MAD) experiments are currently in progress in order to determine the detailed three-dimensional structure of IXT6.

Cloning, Molecular↗
PubMed2005

Crystallization and preliminary X-ray diffraction studies of the pneumococcal teichoic acid phosphorylcholine esterase Pce.

The pneumococcal phosphorylcholine esterase (Pce or CbpE) is a modular protein that hydrolyses the phosphorylcholine residues present in the teichoic and lipoteichoic acids of the pneumococcal cell wall. Pce has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality monoclinic crystals belong to space group C2, with unit-cell parameters a = 169.82, b = 57.26, c = 67.44 A, beta = 112.60 degrees. A 2.7 A resolution SAD data set from a non-isomorphous Gd-HPDO3A Pce derivative was collected at the gadolinium L(III) absorption edge using synchrotron radiation.

Amino Acid Sequence↗
PubMed2005

Structure of a class II TrmH tRNA-modifying enzyme from Aquifex aeolicus.

Biological RNAs contain a variety of post-transcriptional modifications that facilitate their efficient function in the cellular environment. One of the two most common forms of modification is methylation of the 2'-hydroxyl group of the ribose sugar, which is performed by a number of S-adenosylmethionine (SAM) dependent methyltransferases. In bacteria, many of these modifications in tRNA and rRNA are carried out by the alpha/beta-knot superfamily of enzymes, whose SAM-binding pocket is created by a characteristic deep trefoil knot. TrmH, an enzyme found throughout all three kingdoms of life, modifies the universally conserved guanosine 18 position of tRNA. The crystal structure of TrmH from the thermophilic bacterium Aquifex aeolicus has been determined at 1.85 A resolution using data collected from a synchrotron-radiation source. The protein reveals a fold typical of members of the SpoU clan of proteins, a subfamily of the alpha/beta-knot superfamily, with alpha-helical extensions at the N- and C-termini that are likely to be involved in tRNA binding.

Bacteria↗
PubMed2005

Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis.

Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4(1)2(1)2, with unit-cell parameters a = 61.96, c = 210.78 A. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 A resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

Acid Phosphatase↗
PubMed1993

Efficient electrode spacing for examining spatial organization during ventricular fibrillation.

Spatial organization has been observed during episodes of ventricular fibrillation (VF) by recording epicardial unipolar electrograms on a grid of electrodes. In such studies, the choice of spacing between electrodes is an important decision, affecting the resolution and the size of the domain to be studied. A basic tenet of sampling theory, the Nyquist criterion, states that an electrode spacing smaller than half the smallest significant wavelength is required to capture the important details of a spatially sampled process. In this paper, we suggest a method to choose a practical interelectrode spacing by examining wavenumber power spectra of high-resolution VF data recorded from a square 11 x 11 array of electrodes spaced 0.28 mm apart. The plaque was sutured on the epicardium near the left ventricular apex in seven anesthetized pigs. VF was induced with ac simulation. Unipolar extracellular electrograms were simultaneously recorded from each channel for 2 s after the induction of VF. Each signal was sampled in time at 1000 Hz. Wavenumber power spectra were calculated for 100 ms segments using the zero-delay wavenumber spectrum method, for a total of 140 power spectra. All spectra had dominant peaks at the origin and fell off rapidly with increasing wavenumber (decreasing wavelength). In all the spectra, every wavelength shorter than 1.4 mm contributed insignificant power. Furthermore, in 134 of 140 spectra (96%), insignificant power levels were associated with every wavelength shorter than 2.8 mm. These results suggest that, for unipolar extracellular electrodes, an intersensor spacing on the order of 1 mm is appropriate to study organization during early VF.

Animals↗
PubMed2005

High-resolution analysis of ambulatory electrocardiograms to detect possible mechanisms of premature ventricular beats.

For generations of electrocardiogram (ECG) analysis, the presence of premature ventricular beats (PVBs) has been characterized as a common event in the ECG without regard to the mechanism which has caused the PVB in the first place. At best, the coupling interval with the preceding sinus beat may be noted. This viewpoint persisted throughout the era of automated ECG analysis, as well as influencing the treatment of more life threatening events by PVB suppression strategies alone. This study proposed three hypotheses which would link the PVB to a specific mechanism or milieu. Each of these hypotheses requires significant signal processing of the continuously recorded high resolution ECG. Data are presented which demonstrate that abnormal intra-QRS potentials may be linked to a reentrant mechanism for the PVBs and that many patients have significant changes in these potentials in the sinus beats preceding the PVB. Changes in the characteristics of the repolarization as measured in the T/U wave period were also observed and could be linked to triggered activity mechanism for some PVBs. Finally, the role of subclinical ST segment changes also indicates that low grade ischemia may play a role in modulating either PVB mechanism. The data generated by this study suggest that a new view toward PVB mechanism as measured by ECG characteristics may warrant a more rational approach to renewed interest identifying the malignant PVBs and their eventual clinical management.

Algorithms↗
PubMed2004

Artifact analysis and reconstruction improvement in helical cardiac cone beam CT.

With the introduction of cone beam (CB) scanners, cardiac volumetric computed tomography (CT) imaging has the potential to become a noninvasive imaging tool in clinical routine for the diagnosis of various heart diseases. Heart rate adaptive reconstruction schemes enable the reconstruction of high-resolution volumetric data sets of the heart. Artifacts, caused by strong heart rate variations, high heart rates and obesity, decrease the image quality and the diagnostic value of the images. The image quality suffers from streak artifacts if suboptimal scan and reconstruction parameters are chosen, demanding improved gating techniques. In this paper, an artifact analysis is carried out which addresses the artifacts due to the gating when using a three-dimensional CB cardiac reconstruction technique. An automatic and patient specific cardiac weighting technique is presented in order to improve the image quality. Based on the properties of the reconstruction algorithm, several assessment techniques are introduced which enable the quantitative determination of the cycle-to-cycle transition smoothness and phase homogeneity of the image reconstruction. Projection data of four patients were acquired using a 16-slice CBCT system in low pitch helical mode with parallel electrocardiogram recording. For each patient, image results are presented and discussed in combination with the assessment criteria.

Algorithms↗
PubMed2004

Inadequacy of standard screen resolution for localization of seizures recorded from intracranial electrodes.

Seizures recorded during long-term monitoring with implanted intracranial electrodes are typically interpreted by visual inspection alone by using digital display systems. When high-frequency activity is digitized and displayed on a typical monitor, it is altered in ways that are not always appreciated and that may have an impact on the intracranial EEG (ICEEG) interpretation. We describe a case of a neocortical-onset seizure in which false localization occurred with a 12-s per screen display. Because frequencies in excess of 100 Hz are not uncommon in neocortical seizures, at most 4 to 5 s of EEG, depending on the screen resolution, data-sampling rate, and other factors, should be displayed at one time during visual interpretation to localize the seizure onset. Alternatively, spectral analysis should be performed on recordings of neocortical seizures to detect high-frequency activity that may be missed on visual inspection.

Adult↗
PubMed1993

Complex of ribonuclease Sa with a cyclic nucleotide and a proposed model for the reaction intermediate.

The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with exo guanosine 2',3'-cyclophosphorothioate has been refined against 0.2-nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2'-GMP complex. The refinement was based on all data over 1.0-0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2',3'-cyclophosphorothioate, which is a thio analogue of the intermediate of the two-step reaction. However, exo guanosine 2',3'-cyclophosphorothioate is bound in a non-functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.

Binding Sites↗
PubMed2025

Genome wide association study of rice agronomical traits and seed ionome with the NARO Open Rice Collection.

To meet the nutritional needs of the rising human population, genetic variants are necessary for the breeding of new cultivars. Rice (Oryza sativa L.) is a staple food for over half of the world's population. Here, we developed a new rice genetic resource, the NARO Open Rice Collection (NRC) with high-resolution genome data. NRC consists of 623 accessions, and approximately 200 accessions are categorized into three major subgroups, categorized as Indica, Japonica, and Aus. In this study, we performed genome-wide association studies (GWAS) for rice heading date, seed shape, and seed ionome using the NRC. Well-known genes related to heading date and seed shape were detected by GWAS using the NRC accessions. Therefore, we concluded that our new rice collection is suitable for GWAS. In addition, GWAS with each subgroup was advantageous for the detection of particular genes. Finally, we performed GWAS for seed ionome with the aim of improving the nutritional properties of rice, as essential minerals for humans, such as iron (Fe) and zinc (Zn), are not sufficient in rice seeds. Our study revealed that OsATL31, a likely ubiquitin E3 ligase, was involved in the control of Fe and Zn contents in seeds.

Oryza↗
PubMedDate not supplied

Three-dimensional optical coherence tomography of the embryonic murine cardiovascular system.

Optical coherence tomography (OCT) is an emerging high-resolution real-time biomedical imaging technology that has potential as a novel investigational tool in developmental biology and functional genomics. In this study, murine embryos and embryonic hearts are visualized with an OCT system capable of 2-microm axial and 15-microm lateral resolution and with real-time acquisition rates. We present, to our knowledge, the first sets of high-resolution 2- and 3-D OCT images that reveal the internal structures of the mammalian (murine) embryo (E10.5) and embryonic (E14.5 and E17.5) cardiovascular system. Strong correlations are observed between OCT images and corresponding hematoxylin- and eosin-stained histological sections. Real-time in vivo embryonic (E10.5) heart activity is captured by spectral-domain optical coherence tomography, processed, and displayed at a continuous rate of five frames per second. With the ability to obtain not only high-resolution anatomical data but also functional information during cardiovascular development, the OCT technology has the potential to visualize and quantify changes in murine development and in congenital and induced heart disease, as well as enable a wide range of basic in vitro and in vivo research studies in functional genomics.

Animals↗