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At least 955 records · Page 53Linked to original sources
PubMed2000

Crystallization and preliminary X-ray studies of membrane-associated Escherichia coli dihydroorotate dehydrogenase.

Dihydroorotate dehydrogenases (DHODs) are flavin-containing enzymes which catalyse the conversion of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides. Two major families of DHODs have now been identified based on their amino-acid sequence similarities. The two families differ in their reaction mechanisms, but structures are only known of enzymes belonging to family 1. DHOD from Escherichia coli is a typical member of family 2, which contains the membrane-associated enzymes from Gram-negative bacteria and eukaryotes. Yellow crystals grown of this enzyme belong to the space group P4(1)2(1)2 or P4(3)2(1)2. The unit-cell parameters are a = b = 119.2, c = 294.3 A. Owing to the rather large c axis, the currently available resolution of data is 2.2 A.

Cell Membrane↗
PubMed2000

Crystallization and preliminary X-ray analysis of Clostridium botulinum neurotoxin type B.

Single crystals of Clostridium botulinum neurotoxin type B have been obtained by the vapor-diffusion method. These crystals belong to space group P2(1), with unit-cell parameters a = 76.08, b = 123.11, c = 95.86 A, beta = 113.03 degrees and diffract to at least 1.8 A resolution. Native data have been collected from flash-frozen crystals at the National Synchrotron facility of Brookhaven National Laboratory. These crystals often tend to be non-isomorphic.

Botulinum Toxins↗
PubMed2000

Crystallographic studies of the interaction between the ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena: looking for the elusive ferredoxin molecule.

Ferredoxin-NADP(+) reductase (FNR) and its physiological electron donor ferredoxin (Fd) from the cyanobacterium Anabaena PCC7119 have been co-crystallized. The unit-cell parameters are a = b = 63.72, c = 158.02 A and the space group is P2(1)2(1)2(1). The crystal structure has been solved with 2.4 A resolution synchrotron data by molecular replacement, anomalous dispersion and R(min) search methods. For the computations, the crystal was treated as a merohedral twin. The asymmetric unit contains two FNR molecules and one ferredoxin molecule. The packing of the FNR molecules displays a nearly tetragonal symmetry (space group P4(3)2(1)2), whereas the ferredoxin arrangement is orthorhombic. This study provides the first crystallographic model of a dissociable complex between FNR and Fd.

Anabaena↗
PubMed2001

Crystallization and preliminary X-ray analysis of Borrelia burgdorferi outer surface protein C (OspC).

Single crystals of the outer surface protein C (OspC) from Borrelia burgdorferi HB19 have been obtained by the vapor-diffusion method. These crystals belong to space group P2(1), with unit-cell parameters a = 66.218, b = 46.113, c = 112.079 A, beta = 99.30 degrees, and diffract to at least 2.2 A resolution. Native data have been collected from flash-frozen crystals at the National Synchrotron facility of Brookhaven National Laboratory. There are two dimers per asymmetric unit, related by a non-crystallographic twofold axis and a pseudo-translational symmetry.

Amino Acid Sequence↗
PubMed2001

De novo structure determination of vancomycin aglycon using the anomalous scattering of chlorine.

The crystal structure of vancomycin aglycon has been determined by exploiting the anomalous scattering of Cl atoms present within the molecule. Real-space-reciprocal-space cycling with Shake-and-Bake successfully located the chlorine positions from the Bijvoet differences, even though the anomalous difference Patterson map proved to be uninterpretable. The chlorine anomalous differences lacked sufficient phasing power to produce interpretable electron-density maps. However, when combined with high-resolution native data, the chlorine positions were sufficient to determine the structure using either Shake-and-Bake or a tangent-formula expansion.

Anti-Bacterial Agents↗
PubMed2001

Structure of mouse L-chain ferritin at 1.6 A resolution.

Cubic F432 crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique with ammonium sulfate and cadmium sulfate as precipitants. The structure was refined to 2.1 and 1.6 A resolution from data obtained at room temperature and under cryogenic conditions, respectively. The structure of an eight-amino-acid loop insertion in the mouse sequence is found to be highly disordered both at room temperature and at low temperature.

Animals↗
PubMed2002

Generating isomorphous heavy-atom derivatives by a quick-soak method. Part I: test cases.

Screening for heavy-atom derivatives remains a time-consuming and cumbersome process that often results in non-isomorphous derivatives whose phases cannot be combined. Using lysozyme and FcgammaRIII receptor crystals as test cases, an improved soaking method for the generation of conventional heavy-atom derivatives has been developed. The method is based on soaking crystals in heavy-atom compounds for a very brief time at near-saturation concentrations. Compared with the current heavy-atom soaking method, which often takes days to achieve a derivatization, the quick-soak method completes a derivatization within 10 min to 2 h. The bound heavy-atom sites display higher peak heights from quick soaks than from overnight soaks in all cases tested. The quick-soak derivatives also preserved native-like diffraction resolution and data quality that was better than the prolonged-soak derivatives. Furthermore, derivatives generated by brief soaks are more isomorphous to the native than those generated by overnight soaks. Short soaks not only increase the likelihood of success in heavy-atom screening by reducing the pitfalls associated with prolonged soaks, such as lack of isomorphism and overall lattice disorder, but also have the potential to transform a time-consuming derivative screening into an 'on-the-fly' real-time derivatization process.

Animals↗
PubMed2002

Crystallization and preliminary X-ray characterization of archaeal group II chaperonin alpha-subunit from Thermococcus strain KS-1.

The archaeal group II chaperonin from Thermococcus strain KS-1 is composed of two kinds of subunits (alpha and beta). Each of the recombinant subunits was individually expressed in Escherichia coli and purified as homo-hexadecamers of each subunit. Both homo-oligomers facilitate the refolding of denatured proteins in vitro in an ATP-dependent manner. A mutant alpha-subunit homo-oligomer with two amino-acid substitutions, which has the ability to capture the unfolded protein but lacks the ability to refold the unfolded protein, was crystallized in two different conditions. One crystal form was obtained from a high-concentration solution of ammonium sulfate and grew to maximum dimensions of 0.15 x 0.15 x 0.4 mm. The crystals of this form belonged to the tetragonal space group P42(1)2, with unit-cell parameters a = b = 209.3, c = 156.1 A, and diffracted X-rays to 2.4 A resolution with synchrotron radiation. The other form was crystallized from a polyethylene glycol 6000 solution and belonged to the tetragonal space group, with unit-cell parameters a = b = 220.8, c = 182.4 A. This form only diffracts X-rays to 6 A resolution. Diffraction data collected from the former crystal enabled initial successful phases to be obtained by the molecular-replacement method.

Archaeal Proteins↗
PubMed2002

Crystallization of Pichia pastoris lysyl oxidase.

A copper-containing amine oxidase (PPLO) from the yeast Pichia pastoris has been purified and crystallized in two forms. PPLO is a glycoprotein. The molecular mass from SDS-polyacrylamide gels is 112 kDa, consistent with 20% glycosylation by weight (the calculated molecular weight of the polypeptide is 89.7 kDa). Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 163.7, b = 316.1, c = 84.0 A, diffract to 2.65 A resolution. Monoclinic crystals belonging to space group C2, with unit-cell parameters a = 248.4, b = 121.1, c = 151.8 A, beta = 124.6 degrees, diffract to 1.65 A resolution. Native data have been recorded from each crystal form at 100 K using synchrotron radiation. A self-rotation function for the monoclinic crystal form reveals the presence of a non-crystallographic twofold axis perpendicular to the crystallographic twofold axis, consistent with the presence of two dimers in the asymmetric unit.

Crystallization↗
PubMed2003

Structure of 20K endoglucanase from Melanocarpus albomyces at 1.8 A resolution.

The crystal structure of the 20K endoglucanase from the thermophilic fungus Melanocarpus albomyces (Ma20k) has been determined. The structure was refined to 1.8 A resolution using data obtained at 120 K. Ma20k belongs to glycoside hydrolase family 45. The three-dimensional structures of endoglucanase V (EGV) from the fungus Humicola insolens and of an endoglucanase from H. grisea var. thermoidea have previously been determined. The overall structure of Ma20k consists of a six-stranded beta-barrel domain similar to that found previously in family 45 endoglucanases. The flexible loop between strands V and VI, which was disordered in the uncomplexed structures of the Humicola endoglucanases but was ordered in complexed structures of EGV, is found to be well ordered in the native structure of Ma20k. The structure of Ma20k allows comparison between thermophilic and mesophilic proteins of family 45 and different principles for thermostability are discussed.

Amino Acid Sequence↗
PubMed2003

Purification, crystallization and preliminary X-ray analysis of immunogenic virus-like particles formed by infectious bursal disease virus (IBDV) structural protein VP2.

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. VP2 protein, which consists of 452 amino-acid residues, is the primary immunogen of IBDV and contains the epitopes responsible for eliciting neutralizing antibodies. When the chimeric VP2 protein (rVP2H) of a local IBDV strain P3009 was expressed alone using the baculovirus system, virus-like particles of approximately 23 nm in diameter formed spontaneously. Highly pure rVP2H particles, obtained using ammonium sulfate precipitation, immobilized metal-ion affinity chromatography and gel-filtration chromatography, were successfully crystallized using the vapour-diffusion method. These crystals, with a maximum dimension of 0.4 mm, diffracted X-rays to 4.5 A resolution, but data were only collected to 6 A. Preliminary analysis of the diffraction data showed that the rVP2H crystals belong to the cubic space group P2(1)3, with unit-cell parameter 323.1 A. The icosahedral symmetry of the particles is clearly seen in the self-rotation function maps, with dyads and triads coincident with the crystallographic axes. Each asymmetric unit contains 1/3 of the particle, or 20 rVP2H subunits, and there are four particles in a unit cell, probably in a tetrahedral arrangement.

Baculoviridae↗
PubMed2004

Crystallization of a Golgi-associated PR-1-related protein (GAPR-1) that localizes to lipid-enriched microdomains.

The human Golgi-associated PR-1-related protein (GAPR-1) is closely related to plant pathogenesis-related (PR-1) proteins, which are upregulated in response to pathogen attack. Family members have been identified in a variety of organisms, together constituting the superfamily of PR-1 proteins. GAPR-1 is found within lipid-enriched microdomains on the cytosolic side of the endomembrane system. GAPR-1 is tightly anchored to membranes and absent from the cytosol, although it does not possess a membrane-spanning domain. Crystals of recombinantly expressed GAPR-1 have been grown that diffract to high (1.5 A) resolution. Complete data sets have been collected on a trigonal crystal form (P3(1)21/P3(2)21), with unit-cell parameters a = b = 73.5, c = 63.2 A. Molecular replacement using the NMR coordinates of tomato pathogenesis-related protein (28% identity) was unsuccessful and a search for heavy-metal derivatives or alternative phasing methods has been initiated.

Cloning, Molecular↗
PubMed1994

Crystallization and preliminary crystallographic analysis of galactose-1-phosphate uridylyltransferase from Escherichia coli.

Galactose-1-phosphate uridylyltransferase catalyzes the formation of UDP-galactose during normal cellular metabolism, making it an essential enzyme in all cells. The enzyme from Escherichia coli has been crystallized at pH 5.9 in the presence of phenyl-UDP (P(1)-5'-uridyl-P(2)-phenyl diphosphate), a substrate analog, using PEG 10 000 in combination with Li(2)SO(4) and NaCl. Crystals belong to space group P2(1)2(1)2 with unit-cell dimensions a = 58.6, b = 217.6 and c = 69.6 A. There is one dimer or two subunits in the asymmetric unit. Crystals are relatively insensitive to X-ray radiation and diffract beyond 2.5 A resolution. A low-resolution native data set has been recorded.

Journal Article↗
PubMed1996

Studies of monoclinic hen egg-white lysozyme. IV. X-ray refinement at 1.8 A resolution and a comparison of the variable regions in the polymorphic forms.

Monoclinic crystals of hen egg-white lysozyme (E.C. 3.2.1.17, HEL) grown at low pH in the presence of NaNO(3) belong to space group P2(1) with unit-cell dimensions, a = 28.0, b = 62.5, c = 60.9 A and beta= 90.8 degrees with two molecules in the asymmetric unit. 1.8 A resolution intensity data, collected on a CAD-4 diffractometer, contained 17 524 reflections with F > 3sigma (93% complete). Our earlier preliminary 1.8 A model was refitted and refined using X-PLOR to an R value of 0.189. The deviations in the model from ideal geometry are 0.013 A in bond lengths and 2.8 degrees in bond angles. The r.m.s. deviation in the backbone atoms between the two molecules is 0.42 A. A comparison of HEL in different polymorphic crystal forms reveals that the prominent structural variability among them resides in two exposed regions 45-50 and 65-73 which are also regions of lattice contacts.

Journal Article↗
PubMed1996

Structure of bovine eye lens gammaD (gammaIIIb)-crystallin at 1.95 A.

The crystal structure of bovine lens gammaIIIb-crystallin at 2.5 A resolution previously reported was interpreted using a consensus sequence derived from related vertebrate sequences on the assumption that gammaIIIb-crystallin derived from the gammaC-crystallin gene. It has recently been shown that gammaIIIb is a product of the bovine gammaD gene. The structure of gammaIIIb has now been refined with the bovine gammaD sequence using new 1.95 A resolution synchrotron data. The crystallographic R factor was 20.4% for all 33 104 reflection data between 8.0 and 1.95 A measured at 277(1) K. The electron density fully supported the assignment of the gammaD sequence to gammaIIIb. The crystal belongs to space group P2(1)2(1)2(1) with two molecules of molecular mass 20 749 Da in the asymmetric unit in which 219 water molecules were located. The two-domain four-Greek-key motif highly symmetrical protein is very similar in structure to gammaB-crystallin (81% sequence identity). There is a single amino-acid deletion in gammaD in the linker region connecting the two domains. The intermolecular oganization in the crystal lattice is quite different from gammaB as a result of key mutations involving surface residues Leu51, Ile103 and His155. These point mutations will contribute to the intermolecular behaviour of the gamma-crystallins in the eye lens, where they are major components of the densely packed, high refractive index regions of the lens.

Journal Article↗
PubMed1998

The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein component: crystallization and preliminary X-ray analysis.

The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein subunit was crystallized using the hanging-drop technique, with PEG 4000 as precipitant. The crystals belong to space group P3112 or enantiomorph, with unit-cell parameters a = b = 171.0, c = 152.1 A. A solvent content of 75% was determined by a calibrated tetrachloromethane/toluene gradient which corresponds to three monomers per asymmetric unit. A 3 A resolution native data set was collected at beamline W32 of LURE, Orsay, France.

Bacterial Proteins↗
PubMed1998

Preliminary X-ray analysis of a new crystal form of the vanadium-dependent bromoperoxidase from Corallina officinalis.

A new crystal form of the vanadium-dependent bromoperoxidase from Corallina officinalis has been obtained. The crystals exhibit a 'teardrop' morphology and are grown from 2 M ammonium dihydrogen phosphate pH and diffract to beyond 1.7 A resolution. They are in tetragonal space group P4222 with unit-cell dimensions of a = b = 201.9, c = 178.19 A, alpha = beta = gamma = 90 degrees. A 2.3 A resolution native data set has been collected at the Hamburg Synchrotron. A mercury derivative data set has also been collected, and the heavy-atom positions have been determined. The self-rotation function and the positions of the heavy atoms are consistent with the molecule being a dodecamer with local 23 symmetry.

Crystallography, X-Ray↗
PubMed1998

Crystallization and preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from Sacchromyces cerevisiae (baker's yeast).

Two related and not thoroughly resolved issues in biochemistry concern the role, if any, of enzyme surfaces in routine metabolism and the method by which metabolic intermediates move between enzyme active sites during multi-step degradation or synthesis. An important enzyme for which a detailed three-dimensional structural analysis has been initiated is yeast glyceraldehyde 3-phosphate dehydrogenase (yGAP-DH). This enzyme is active as a tetramer of total molecular weight of 145 kDa and requires nicotinamide adenine dinucleotide (NAD+) as cofactor. In this report, the crystallization and preliminary crystallographic characterization of several crystal forms of yGAP-DH are described. Of the five distinct crystal forms, the most suitable was found to contain the holo-enzyme, and the crystals were grown by the vapor-diffusion method using polyethylene glycol 6000 as precipitant, sodium acetate as buffer (pH 4.6), and NAD+ and dithiothreitol as additives. The crystals belong to the orthorhombic space group P21212, with cell dimensions of a = 87.33, b = 96.11 and c = 115.34 A. These crystals are mechanically strong, relatively stable in the X-ray beam and diffract X-rays (from a normal rotating-anode radiation source) to better than 2 A resolution. A full 2.1 A resolution diffraction data set (98% completion) has been measured. The three-dimensional structures of related GAP-DH enzymes from several other sources have been determined and reported, and are available for a molecular replacement structure solution.

Binding Sites↗