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At least 181 records · Page 10Linked to original sources
PubMed2000

Crystallization and preliminary X-ray crystallographic analysis of thioesterase I from Escherichia coli.

The Escherichia coli thioesterase I specifically catalyzes the deacylation of fatty acyl-CoA thioesters, especially those with long acyl groups (C(12)-C(18)). Single crystals of thioesterase I (E.C. 3. 1.2.2) from E. coli have been obtained using methoxypolyethylene glycol 5000 (PEG-MME 5K) as a precipitant at room temperature in 21 d. The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2, with unit-cell parameters a = b = 50.85 (7), c = 171.5 (1) A. The crystals diffract to beyond 2.4 A resolution. There is one molecule of molecular weight 20.5 kDa in the asymmetric unit, with a solvent content of 55%.

Amino Acid Motifs↗
PubMed2003

Crystallization and preliminary X-ray diffraction analysis of thermophilic imidase from pig liver.

Imidase is an enzyme, also known as dihydropyrimidinase (EC 3.5.2.2), hydantoinase, dihydropyrimidine hydrase or dihydropyrimidine amidohydrolase, that catalyzes the reversible hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate and many other imides. Substrate specificity, metal content and amino-acid sequence all differ significantly between bacterial and mammalian imide-hydrolyzing enzymes. In this study, a thermophilic imidase was isolated from pig liver and crystallized. Two kinds of imidase crystals were grown by the hanging-drop vapour-diffusion method using polyethylene glycol MME 5000 and 2-propanol as precipitants. One belongs to the triclinic P(1) space group, with unit-cell parameters a = 96.35, b = 96.87, c = 154.87 A, alpha = 82.10, beta = 72.54, gamma = 77.19 degrees, and the other belongs to the orthorhombic C222(1) space group, with unit-cell parameters a = 113.92, b = 157.22, c = 156.21 A.

Amidohydrolases↗
PubMed2003

Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase.

Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B. subtilis. The enzyme was purified in two steps to homogeneity prior to crystallization. Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions. The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0. They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A. A complete data set was collected to 2 A from a single native crystal using synchrotron radiation.

Bacillus subtilis↗
PubMed2003

Purification, crystallization and preliminary X-ray diffraction data of L7Ae sRNP core protein from Pyrococcus abyssii.

The L7Ae sRNP core protein from Pyrococcus abyssii was crystallized using the sitting-drop vapour-diffusion method. Crystals were obtained in the presence of MgCl(2), PEG 2000 MME and acetate buffer at pH 4.0. A native data set has been collected at 2.9 A resolution using a rotating-anode generator at room temperature. Crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 70.7, b = 112.9, c = 34.8 A. There are two monomers of MW 14 200 Da per asymmetric unit and the packing density V(M) is 2.45 A(3) Da(-1). A molecular-replacement analysis gave solutions for the rotation and translation functions.

Archaeal Proteins↗
PubMed2004

Purification, crystallization and preliminary X-ray studies of sylvaticin, an elicitin-like protein from Pythium sylvaticum.

Sylvaticin belongs to the elicitin family. These 10 kDa oomycetous proteins induce a hypersensitive response in plants, including necrosis and cell death, but subsequently leading to a non-specific systemic acquired resistance (SAR) against other pathogens. Sylvaticin has been crystallized using PEG 2000 MME as a precipitant agent in the presence of nickel chloride. The crystals belong to space group C2, with unit-cell parameters a = 99.29, b = 25.67, c = 67.45 A, beta = 99.66 degrees. Diffraction data were recorded to 2.1 A resolution at a synchrotron-radiation source.

Algal Proteins↗
PubMed2004

Crystallization and preliminary X-ray diffraction analysis of the hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum.

NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum was crystallized in the apo- and holoenzyme forms. Crystals were obtained using 2-propanol and polyethylene glycol MME 550 as precipitants for the apoenzyme and holoenzyme, respectively. The apoenzyme crystals belong to the trigonal space group P3(1)21 or its enantiomorph P3(2)21. The asymmetric unit contains three subunits; the values of the Matthews coefficient (VM) and the solvent content are 2.9 A3 Da-1 and 57%, respectively. A native data set was collected to a highest resolution limit of 4.0 A on an in-house X-ray source using a rotating-anode generator (overall Rsym of 12.3% and completeness of 97%). The holoenzyme crystals belong to the orthorhombic space group P2(1)2(1)2(1); the asymmetric unit contains one hexamer, giving a VM of 2.79 A3 Da-1 and a solvent content of 55%. Native and derivative data sets were collected. The crystals diffract to a maximum resolution of 2.8 A on the KEK-NW12 beamline at the Photon Factory and gave a data set with an overall Rsym of 7.9% and a completeness of 91%. Attempts are being made to solve the structure by the SIRAS method.

Archaeal Proteins↗
PubMed2004

Purification, crystallization and preliminary X-ray analysis of Methanococcus jannaschii TATA box-binding protein (TBP).

TATA box-binding protein (TBP) from Methanococcus jannaschii has been crystallized by the hanging-drop vapour-diffusion method using PEG MME 2000 as a precipitant. The crystal belongs to space group P21, with unit-cell parameters a = 53.2, b = 55.5, c = 123.4 A,a = 90.0, fi = 91.0, y = 90.0 degrees, and contains four molecules in the asymmetric unit. A data set was collected to 1.9 A resolution using synchrotron radiation. A molecular-replacement solution was found using the structure of TBP from Sulfolobus acidocaldarius as a model. Crystallographic refinement is in progress.

Amino Acid Sequence↗
PubMed2004

Crystallization and preliminary crystallographic analysis of the 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase from the carbazole-degrader Pseudomonas resinovorans strain CA10.

CarBaBb, the class III extradiol dioxygenase involved in carbazole degradation by Pseudomonas resinovorans CA10, was crystallized at 278 K by the hanging-drop vapour-diffusion method using PEG MME 550 as a precipitant. The crystals had a transparent thin square-pillar shape and belonged to space group P2(1)2(1)2, with unit-cell parameters a = 122.8, b = 144.6, c = 49.2 A, alpha = beta = gamma = 90 degrees . The crystals diffracted to a maximum resolution of 1.9 A and gave a data set with an overall R(merge) of 5.7% and a completeness of 98.6%. The V(M) value was 2.52 A(3) Da(-1), which indicated a solvent content of 51.2%.

Crystallization↗
PubMed2004

Crystallization and preliminary X-ray analysis of the Streptomyces olivaceoviridis NgcE binding protein of the ABC transporter for N-acetylglucosamine.

The NgcE protein binds N-acetylglucosamine (GIcNAc) as well asN,N'-diacetylchitobiose and is a component of the ABC transporter Ngc for GIcNAc uptake in Streptomyces olivaceoviridis. After cloning the corresponding gene in an Escherichia coli host, the NgcE protein was overproduced in a soluble form within the cytoplasm and purified to homogeneity by four consecutive chromatographic processes. Crystals of NgcE that grew in the presence of 1 mM GlcNAc,20%(w/v) PEG MME 2000 and 100 mM Tris-HCI pH 8.5 had a plate-like shape and belonged to either space group P21212 (unit-cell parameters a = 59.9, b = 153.0, c = 41.7 A) or P212121 (a = 58.1, b = 96.3, c = 151.7 A). The former crystals diffracted to 1.8 A resolution andthe latter to 2.2 A. Selenomethionine-containing crystals were generated under the same conditions and belonged to space group P212121 with unit-cell parameters a = 58.4, b = 96.6, c = 152.5 A, and diffracted to 2.0 A resolution.

ATP-Binding Cassette Transporters↗
PubMed1993

Crystallization of a Humicola lanuginosa lipase-inhibitor complex with the use of polyethylene glycol monomethyl ether.

The fungal Humicola lanuginosa lipase complexed with the inhibitor n-dodecylphosphonate ethyl ester was crystallized in space group P2(1)2(1)2(1) with pseudotetragonal unit-cell parameters of a = 131.7 (2), b = 131.3 (1), c = 75.4 (1) A. 92% of X-ray diffraction data to 2.8 A resolution were collected with a final R(merge) = 8.5%. The crystals were grown using a new kind of precipitant - polyethylene glycol monomethyl ether (Peg-mme) of molecular weight 5000.

Journal Article↗
PubMed2005

The purification and crystallization of mDia1 in complex with RhoC.

An N-terminal construct of mouse mDia1 was recombinantly expressed in Escherichia coli, purified and crystallized in complex with truncated human RhoC using the hanging-drop vapour-diffusion method. Crystals were obtained using PEG 2K MME and MgSO4 as a precipitating agent and belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 148.4, b = 85.2, c = 123.2 A. Complete native and SeMet-derivative data sets were collected at 100 K to 3.0 and 3.4 A resolution, respectively, using synchrotron radiation.

Animals↗
PubMed2005

Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 A and belongs to space group P2(1), with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 A, alpha = gamma = 90, beta = 103.8 degrees. Diffraction data for the type I crystal gave an overall Rmerge of 8.0% and a completeness of 100%. Its VM value is 2.63 A3 Da(-1), indicating a solvent content of 53.2%.

Bacterial Proteins↗
PubMed2005

Expression, crystallization and preliminary diffraction studies of the Pseudomonas putida cytochrome P450cam operon repressor CamR.

The Pseudomonas putida cam repressor (CamR) is a homodimeric protein that binds to the camO DNA operator to inhibit the transcription of the cytochrome P450cam operon camDCAB. CamR has two functional domains: a regulatory domain and a DNA-binding domain. The binding of the inducer D-camphor to the regulatory domain renders the DNA-binding domain unable to bind camO. Native CamR and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. Native CamR was crystallized under the following conditions: (i) 12-14% PEG 4000, 50 mM Na PIPES, 0.1 M KCl, 1% glycerol pH 7.3 at 288 K with and without camphor and (ii) 1.6 M P(i), 50 mM Na PIPES, 2 mM camphor pH 6.7 at 278 K. The selenomethionyl derivative CamR did not crystallize under either of these conditions, but did crystallize using 12.5% PEG MME 550, 25 mM Na PIPES, 2.5 mM MgCl2 pH 7.3 at 298 K. Preliminary X-ray diffraction studies revealed the space group to be orthorhombic (P2(1)2(1)2), with unit-cell parameters a = 48.0, b = 73.3, c = 105.7 A. Native and selenomethionyl derivative data sets were collected to 3 A resolution at SPring-8 and the Photon Factory.

Bacterial Proteins↗
PubMed2005

Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2a.

Diadenosine tetraphosphate (Ap4A) hydrolase (EC 3.6.1.41) hydrolyzes Ap4A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap4A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap4A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap4A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap4A hydrolase crystals diffract X-rays to 3.26 A and belong to space group P2(1), with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 A, beta = 95.7 degrees.

Acid Anhydride Hydrolases↗
PubMed2005

Purification, crystallization and preliminary X-ray analysis of the catalytic domain of the Escherichia coli tRNase colicin D.

The tRNase domain of colicin D, which cleaves only tRNA(Arg)s at the 3' side of their anticodon loops, has been expressed in Escherichia coli with its inhibitor protein and purified to a form free from the inhibitor using a low-pH buffer. Crystals were obtained by the hanging-drop vapour-diffusion method at 278 K from a buffer containing 100 mM Tris-HCl pH 8.5, 22% PEG MME 2000 and 1 mM nickel(II) chloride. Diffraction data to 1.05 A resolution were collected at BL41XU, SPring-8. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.7, b = 65.5, c = 96.5 A.

Catalytic Domain↗
PubMed2006

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase.

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 A, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 A resolution using the laboratory X-ray source and are suitable for crystal structure determination.

Animals↗
PubMed2006

Crystallization and preliminary characterization of a highly thermostable lectin from Trichosanthes dioica and comparison with other Trichosanthes lectins.

A lectin from Trichosanthes dioica seeds has been purified and crystallized using 25%(w/v) PEG 2K MME, 0.2 M ammonium acetate, 0.1 M Tris-HCl pH 8.5 and 50 microl 0.5%(w/v) n-octyl beta-D-glucopyranoside as thick needles belonging to hexagonal space group P6(4). Unit-cell parameters were a = b = 167.54, c = 77.42 A. The crystals diffracted to a Bragg spacing of 2.8 A. Both the structures of abrin-a and T. kirilowii lectin could be used as a model in structure determination using the molecular-replacement method; however, T. kirilowii lectin coordinates gave better values of reliability and correlation parameters. The thermal, chemical and pH stability of this lectin have also been studied. When heated, its haemagglutination activity remained unaffected up to 363 K. Other stability studies show that 4 M guanidinium hydrochloride (Gdn-HCl) initiates unfolding and that the protein is completely unfolded at 6 M Gdn-HCl. Treatment with urea resulted in a total loss of activity at higher concentrations of denaturant with no major structural changes. The protein remained stable over a wide pH range, from pH 6 to pH 12, except for partial unfolding at extremely alkaline pH. The role of disulfide bonds in the protein stability was found to be insignificant. Rayleigh light-scattering studies showed no molecular aggregation in any of the extreme treated conditions. The unusual stability of this lectin resembles that of type II ribosome-inactivating proteins (type II RIPs), which is also supported by structure determination. The structural features observed in a preliminary electron-density map were compared with the other two available Trichosanthes lectin structures.

Crystallization↗
PubMed2006

Expression, purification and crystallization of a birnavirus-encoded protease, VP4, from blotched snakehead virus (BSNV).

Blotched snakehead virus (BSNV) is a member of the Birnaviridae family that requires a virally encoded protease known as VP4 in order to process its polyprotein into viral capsid protein precursors (pVP2 and VP3). VP4 belongs to a family of serine proteases that utilize a serine/lysine catalytic dyad mechanism. A mutant construct of VP4 with a short C-terminal truncation was overexpressed in Escherichia coli and purified to homogeneity for crystallization. Using the sitting-drop vapour-diffusion method at room temperature, protein crystals with two distinct morphologies were observed. Cubic crystals grown in PEG 2000 MME and magnesium acetate at pH 8.5 belong to space group I23, with unit-cell parameters a = b = c = 143.8 angstroms. Trigonal crystals grown in ammonium sulfate and glycerol at pH 8.5 belong to space group P321/P312, with unit-cell parameters a = b = 158.2, c = 126.4 angstroms.

Base Sequence↗