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Crystals Suitable (crystal + suitable)
Selected AbstractsCrystallization and preliminary X-ray crystallographic analysis of p24, a component of the potato nuclear factor PBF-2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002Darrell Desveaux The Solanum tuberosum (potato) nuclear factor PBF-2 is implicated in pathogen-induced expression of the pathogenesis-related gene PR-10a. Crystals of the DNA-binding component of PBF-2, p24, have been obtained at 277,K in 20,mM Tris,HCl pH 8.0. Recombinant protein with a His tag at its C-terminus was overexpressed in Escherichia coli in the presence and absence of selenomethionine and was purified using a combination of HiTrap affinity columns and gel-filtration chromatography. Crystals suitable for structural analysis were obtained for both native and selenomethionine-labelled proteins and yielded diffraction data at 100,K that were processed to 2.3 and 2.8,Å resolution, respectively. The p24 protein crystals belong to space group P212121, with unit-cell parameters a = 69.4,(69.1), b = 89.4,(90.5), c = 144.1,(144.3),Å. The asymmetric unit contains four protomers, giving a crystal volume per protein mass (VM) of 2.23,Å3,Da,1 and a solvent content of 45% by volume. [source] Crystallization and preliminary X-ray crystallographic analysis of SMU.412c protein from the caries pathogen Streptococcus mutansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Zhao-Yang Ye The smu.412c gene encodes a putative histidine triad-like protein (SMU.412c) with 139 residues that is involved in cell-cycle regulation in Streptococcus mutans. The gene was cloned into the expression vector pET28a and subsequently expressed in Escherichia coli strain BL21 (DE3) to give a substantially soluble form of SMU.412c with a His6 tag at its N-terminus. The recombinant protein was purified to homogeneity in a two-step procedure involving Ni2+ -chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained using the sitting-drop vapour-diffusion method and diffracted to 1.8,Å resolution on beamline BL6A at Photon Factory, Tsukuba, Japan. The crystal belonged to space group P41212, with unit-cell parameters a = b = 53.5, c = 141.1,Å. [source] Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2005Jun Iwaki Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363,K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5,M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = b = 66.1, c = 196.2,Å, and diffracted to beyond 2.15,Å resolution at 100,K. [source] Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2005Dan Li Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2,,5, RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3,Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77,Å, , = 113.56°. [source] In Quest of the Double Rotaxane Formation of the Bis(coronand) (1,5),(2,4)-Durenetetrayl-bis(18-crown-5) with Organomagnesium CompoundsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 1 2004Gerard P. M. van Klink Abstract The interaction between the title compound, bis(coronand) 8,and the diarylmagnesium compounds Ph2Mg and (p - tBuC6H4)2Mg in diethyl ether leads to the formation of 1:2 complexes (9 and 10, respectively), irrespective of the initial ratio of the components. In [D8]toluene, the three complex types can be discerned by 1H NMR spectroscopy: double side-on (9a, 10a), side-on/rotaxane (9b, 10b), and, to a very minor extent, double rotaxane (9c, 10c). In the case of 10a and 10b, the temperature dependence showed that rotaxane formation is enthalpically favored at the expense of a more negative entropy. As crystals suitable for structure determination could not be obtained from 9 and 10, the interaction of 8 with the sterically less demanding Hg(SCN)2 was studied. In this case, analogous pseudorotaxanes are formed exclusively, and both the 1:2 complex [8·{Hg(SCN)2}2] (11) and its 1:1 analogue 12 were observed. The double rotaxane structure of 11 was confirmed by X-ray crystal structure determination. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Tripodands with Phenyl and Thiophenyl Rings and Nitrogen Bridgehead Atoms,EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 23 2006Martin Baier Abstract The flexible tripodands 7,9 and 15 with phenyl and thiophenyl rings as "legs" and nitrogen as bridgehead atoms have been synthesized by three-component condensation reactions of the corresponding amine with the aryl halide. The more rigid species 10,14 and 17 were built up from the podands 7,9 as well as from their iodine substitution products 33,35 by a sequence of ethynylation and C,C coupling reactions. Podand 16 was prepared from tris-iodide 36 by Sonogashira coupling with phenylacetylene. In the cases of 7, 12, 15,17, 22, 24, 35, 36, and 41 the structural parameters were determined by X-ray studies. With the exception of 7, 12, and 17, all structures show either close intermolecular contacts between heteroatoms (15, 22, 24, 35, and 36), C,H···N hydrogen bonding (41), or are closely packed as a result of ,···, stacking (16). We were able to isolate silver triflate complexes of 9, 10, and 16, and in the case of 9 we obtained crystals suitable for X-ray diffraction studies. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Engineering of a monomeric and low-glycosylated form of human butyrylcholinesteraseFEBS JOURNAL, Issue 2 2002Expression, characterization, crystallization, purification Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 Å resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 Å, c = 124.9 Å, giving a Vm of 2.73 Å3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general. [source] Structural determination of the stable and meta-stable forms of atomoxetine HCl using single crystal and powder X-ray diffraction methodsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2006Gregory A. Stephenson Abstract StratteraÔ is the first FDA-approved nonstimulant medication for the treatment of Attention Deficit Hyperactivity Disorder (ADHD) in children, adolescents, and adults. Two polymorphic forms and an amorphous form of the active pharmaceutical ingredient, atomoxetine HCl, were discovered during drug development. The thermodynamically stable polymorphic form was selected for the commercial product. The stable form readily grows as crystals suitable for single crystal diffraction. The meta-stable crystal form is isolated by rapid crystallization, providing crystals that are too small for routine single crystal methods; consequently its structure was determined by X-ray powder diffraction. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95: 1677,1683, 2006 [source] Evaluation of crystalline objects in crystallizing protein droplets based on line-segment information in greyscale imagesACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2006Kuniaki Kawabata Several automated crystallization systems have recently been developed for high-throughput X-ray structure analysis. However, the evaluation process for the growth state of crystallizing protein droplets has not yet been completely automated. This paper proposes a new evaluation method for crystalline objects in automated crystallization experiments. The main objective is to determine whether a droplet contains crystals suitable for diffraction experiments and analysis. The evaluation method developed here involves extracting line-segment features from an image of the droplet and discriminating the state of crystallization using classifiers based on line features. In order to verify the efficacy of the proposed method, it was used to classify images obtained by an automated crystallization system. [source] Purification, crystallization and preliminary X-ray crystallographic analysis of a cysteine-rich secretory protein (CRISP) from Naja atra venomACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2004Yu-Ling Wang Cysteine-rich secretory proteins (CRISPs) play an important role in the innate immune system and are transcriptionally regulated by androgens in several tissues. The proteins are mostly found in the epididymis and granules of mammals, whilst a number of snake venoms also contain CRISP-family proteins. The natrin protein from the venom of Naja atra (Taiwan cobra), which belongs to a family of CRISPs and has a cysteine-rich C-terminal amino-acid sequence, has been purified using a three-stage chromatography procedure and crystals suitable for X-ray analysis have been obtained using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.58,Å resolution using synchrotron radiation; the crystals belong to space group C2221, with unit-cell parameters a = 59.172, b = 65.038, c = 243.156,Å. There are two protein molecules in the asymmetric unit and the Matthews coefficient is estimated to be 2.35,Å3,Da,1, corresponding to a solvent content of 47.60%. [source] Cloning, purification, crystallization and preliminary crystallographic studies of Bradyrhizobium fucosyltransferase NodZACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2004Krzysztof Brzezinski The ,-1,6-fucosyltransferase NodZ from Bradyrhizobium sp. WM9 (Lupinus), composed of 325 amino acids with a molecular weight of 37,kDa, has been cloned, expressed and purified. Protein crystals suitable for X-ray diffraction were obtained under optimized crystallization conditions using ammonium dihydrogen phosphate as a precipitant. The crystals are hexagonal and belong to space group P6122 or P6522, with unit-cell parameters a = 125.5, c = 95.6,Å, and contain 56.8% solvent and a single protein molecule in the asymmetric unit. Native data were collected to 2.85,Å using synchrotron radiation and cryogenic conditions. The native crystals were soaked in a mother-liquor solution containing 2.5,mM [Ta6Br12]2+ cluster for derivatization and SAD data were collected to 3.4,Å at the tantalum LIII absorption peak. [source] A distinct binding mode of a hydroxyethylamine isostere inhibitor of HIV-1 proteaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2001Jan Dohnálek Crystallization conditions for an HIV-1 protease,inhibitor complex were optimized to produce crystals suitable for X-ray diffraction experiments. The X-ray structure of the HIV-1 protease complex was solved and refined at 3.1,Å resolution. In contrast to Saquinavir, the mimetic hydroxy group of the inhibitor Boc-Phe-,[(S)-CH(OH)CH2NH]-Phe-Glu-Phe-NH2 is placed asymmetrically with respect to the non-crystallographic twofold axis of the protease dimer so that hydrogen bonds between the amino group of the inhibitor and the catalytic aspartates can be formed. The inhibitor binds in the centre of the active site by a compact network of hydrogen bonds to Gly27, Gly127, Asp25, Asp125 and via the buried water molecule W301 to Ile50 and Ile150. [source] Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastorisACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2000Glenn E. Dale Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor. The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris. The protein was purified to homogeneity and single crystals have been obtained. Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP,phosphoramidon binary complex and the apo enzyme. [source] Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoproteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Jens Hausmann Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an ,100,kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0,Å resolution and allowed the collection of complete diffraction data to about 2.6,Å resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed. [source] Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-IACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Xenia Bogdanovi DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P21212. The DppA crystal diffracted X-rays to 1.9,Å resolution using an in-house X-ray generator. [source] Crystallization and preliminary X-ray diffraction analysis of the fructofuranosidase from Schwanniomyces occidentalisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Aitana Polo Schwanniomyces occidentalis invertase is an extracellular enzyme that releases ,-fructose from the nonreducing termini of various ,- d -fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The enzyme has been expressed in Saccharomyces cerevisiae. Recombinant and wild-type forms, which showed different glycosylation patterns, were crystallized by vapour-diffusion methods. Although crystallization trials were conducted on both forms of the protein, crystals suitable for X-ray crystallographic analyses were only obtained from the wild-type enzyme. The crystals belonged to space group P212121, with unit-cell parameters a = 105.78, b = 119.49, c = 137.68,Å. A diffraction data set was collected using a synchrotron source. Self-rotation function and sedimentation-velocity experiments suggested that the enzyme was dimeric with twofold symmetry. [source] Protein preparation, crystallization and preliminary crystallographic studies of Bacillus subtilis glycinamide ribonucleotide transformylaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Yu-He Liang Glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (FTHF) to glycinamide ribonucleotide (GAR), which is an essential step in the de novo synthesis pathway of purines. In Bacillus subtilis, GART is encoded by the gene purN. In order to study the structure and function of B. subtilis GART, the purN gene was amplified, cloned into an expression vector and expressed in soluble form in Escherichia coli. The protein was purified to homogeneity and crystals suitable for X-ray data collection were obtained. These crystals diffracted to 2.5,Å resolution and belonged to space group P3121, with unit-cell parameters a = b = 95.5, c = 64.0,Å. [source] Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Didem Sutay Kocabas Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8,Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P21 and contained one tetramer per asymmetric unit. [source] Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia boliviana Piper seedsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Tales Rocha Moura Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293,K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1,M HEPES pH 7.5 and 3.0,M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99,Å, , = 90.0, , = 120.8, , = 90.0°. Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5,Å resolution. [source] Expression, purification, crystallization and preliminary crystallographic analysis of the proliferation-associated protein Ebp1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2007Eva Kowalinski ErbB-3-binding protein 1 (Ebp1) is a member of the family of proliferation-associated 2G4 proteins (PA2G4s) and plays a role in cellular growth and differentiation. Ligand-induced activation of the transmembrane receptor ErbB3 leads to dissociation of Ebp1 from the receptor in a phosphorylation-dependent manner. The non-associated protein is involved in transcriptional and translational regulation in the cell. Here, the overexpression, purification, crystallization and preliminary crystallographic studies of Ebp1 from Homo sapiens are reported. Initially observed crystals were improved by serial seeding to single crystals suitable for data collection. The optimized crystals belong to the tetragonal space group P41212 or P43212 and diffracted to a resolution of 1.6,Å. [source] Preliminary X-ray diffraction analysis of the cytoplasmic N-terminal domain of the Na/HCO3 cotransporter NBCe1-AACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2006Harindarpal S. Gill The N-terminal cytoplasmic domain of the Na+ -coupled HCO cotransporter NBCe1-A (NtNBCe1) has been linked with proximal renal tubular acidosis. In a previous purification study of recombinant NtNBCe1, crystal growth at a suboptimal protein concentration (<1,mg,ml,1) yielded small single diamond-shaped crystals that diffracted poorly. In the present study, by increasing the protein concentration 50-fold, the crystal size was doubled and robustness was also improved. Crystal annealing made the crystals suitable for X-ray diffraction. The crystals either belong to space group P3121 or P31 with pseudo P3121 symmetry, with unit-cell parameters a = 51.7, b = 51.7, c = 200.6,Å, , = , = 90, , = 120°, and diffract X-rays to 3.0,Å resolution. The calculated Matthews number is 1.9,Å3,Da,1, with two monomers of molecular weight ,83,kDa in the asymmetric unit. The molecular- replacement packing solution shows that the molecules form dimers by a domain-swapping mechanism. [source] Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallizationACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2005David M. Anstrom Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success. [source] Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2005Oliver Einsle Acetylene hydratase is a tungsten-containing hydroxylase that converts acetylene to acetaldehyde in a unique reaction that requires a strong reductant. The subsequent disproportionation of acetaldehyde yields acetate and ethanol. Crystals of the tungsten/iron,sulfur protein acetylene hydratase from Pelobacter acetylenicus strain WoAcy 1 (DSM 3246) were grown by the vapour-diffusion method in an N2/H2 atmosphere using polyethylene glycol as precipitant. Growth of crystals suitable for X-ray analysis strictly depended on the presence of TiIII citrate or dithionite as reducing agents. [source] Crystallization and preliminary X-ray diffraction studies of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2005TAE12 An NAD+ -dependent psychrophilic alcohol dehydrogenase (ADH) from the Antarctic psychrophile Moraxella sp. TAE123 has been purified to homogeneity. The enzyme consists of four identical subunits, each containing two Zn ions. Protein crystals suitable for X-ray diffraction were obtained under optimized salting-out crystallization conditions using ammonium sulfate as a precipitating agent. The crystals are hexagonal bipyramids and belong to space group P3121 or P3221, with unit-cell parameters a = 136.4, c = 210.7,Å. They contain one protein homotetramer in the asymmetric unit. Diffraction data were collected to 2.2,Å under cryogenic conditions using synchrotron radiation. [source] | ||||||||||