scholarly journals Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin from Artocarpus incisa seeds

2015 ◽  
Vol 71 (10) ◽  
pp. 1282-1285 ◽  
Author(s):  
Ana Cristina de Oliveira Monteiro-Moreira ◽  
Humberto D'Muniz Pereira ◽  
Antonio Eufrasio Vieira Neto ◽  
Frederico Bruno Mendes Batista Moreno ◽  
Marina Duarte Pinto Lobo ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Spectrometric Analysis ◽  
Carbohydrate Binding ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Solution ◽  
Artocarpus Incisa

Frutalin is an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and is a powerful tool for tumour biomarker discovery. The crystallization and preliminary X-ray diffraction analysis of this lectin, which was isolated from Artocarpus incisa seeds, are reported here. Frutalin was purified and submitted to mass-spectrometric analysis. Diverse masses at approximately 16 kDa were observed in the deconvoluted spectra, which support the presence of isoforms. The best frutalin crystals were grown within a week in 0.1 M citric acid pH 3.5 which contained 25% PEG 3350 as a precipitant at 293 K, and diffracted to a maximum resolution of 1.81 Å. The monoclinic crystals belonged to space group I2, with unit-cell parameters a = 76.17, b = 74.56, c = 118.98 Å, β = 96.56°. A molecular-replacement solution was obtained which indicated the presence of four monomers per asymmetric unit. Crystallographic refinement of the structure is in progress.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosa, a putative reductase participating in aeruginosin biosynthesis

2015 ◽  
Vol 71 (4) ◽  
pp. 466-470 ◽  
Author(s):  
Ruyi Ding ◽  
Cui Xu ◽  
Xu Chen ◽  
Mengyun Bao ◽  
Xiaoting Qiu
Keyword(s):  
Diffraction Analysis ◽  
Biosynthetic Pathway ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Antithrombotic Activity ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method

The 2-carboxy-6-hydroxyoctahydroindole moiety is an essential residue for the antithrombotic activity of aeruginosins, which are a class of cyanobacteria-derived bioactive linear tetrapeptides. The biosynthetic pathway of the 2-carboxy-6-hydroxyoctahydroindole moiety has not yet been resolved. AerF was indicated to be involved in the biosynthesis of the 2-carboxy-6-hydroxyoctahydroindole moiety. This study reports the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosawith a C-terminal His6tag. The crystal diffracted to a maximum resolution of 1.38 Å and belonged to the tetragonal space groupP4322, with unit-cell parametersa=b= 101.581,c= 116.094 Å. The calculated Matthews coefficient and solvent content of the crystal were 2.47 Å3 Da−1and 50.32%, respectively. The initial model of the structure was obtained by the molecular-replacement method and refinement of the structure is in progress.

scholarly journals Cloning, expression, purification, crystallization and X-ray crystallographic analysis of (S)-3-hydroxybutyryl-CoA dehydrogenase fromClostridium butyricum

2014 ◽  
Vol 70 (4) ◽  
pp. 485-488
Author(s):  
Eun-Jung Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Crystal Volume

(S)-3-Hydroxybutyryl-CoA dehydrogenase fromClostridium butyricum(CbHBD) is an enzyme that catalyzes the second step in the biosynthesis ofn-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. TheCbHBD protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 2 Mammonium sulfate, 0.1 MCAPS pH 10.5, 0.2 Mlithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space groupR3, with unit-cell parametersa=b= 148.5,c= 201.6 Å. With four molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 3.52 Å3 Da−1, which corresponds to a solvent content of approximately 65.04%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophileHalothermothrix orenii

2012 ◽  
Vol 68 (2) ◽  
pp. 240-243 ◽  
Author(s):  
Lokesh D. Kori ◽  
Andreas Hofmann ◽  
Bharat K. C. Patel
Keyword(s):  
Metal Ion ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Metal Ion Affinity Chromatography

A ribokinase gene (rbk) from the anaerobic halothermophilic bacteriumHalothermothrix oreniiwas cloned and overexpressed inEscherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 45.6,b= 61.1,c= 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

scholarly journals Crystallization and preliminary X-ray analysis of a highly stable novel SGNH hydrolase (Est24) fromSinorhizobium meliloti

2014 ◽  
Vol 70 (2) ◽  
pp. 193-195 ◽  
Author(s):  
Bum Han Ryu ◽  
Duy Duc Nguyen ◽  
Tri Duc Ngo ◽  
Changsuk Oh ◽  
Ramesh Pandian ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Structure Refinement ◽  
Ammonium Phosphate ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Hydrolysis Of ◽  
Hydrolase Family

The SGNH hydrolase family includes enzymes that catalyze the hydrolysis of a broad range of substrates. Here, the crystallization and preliminary X-ray crystallographic studies of a novel SGNH hydrolase (Est24) fromSinorhizobium melilotiwere performed. Recombinant Est24 protein containing an N-terminal His tag was expressed inEscherichia coliand purified to homogeneity. Est24 was then crystallized using a solution consisting of 0.2 Mammonium phosphate pH 4.6, 20% polyethylene glycol 3350. X-ray diffraction data were collected to a resolution of 1.45 Å with anRmergeof 9.4%. The Est24 crystals belonged to space groupC2, with unit-cell parametersa= 129.09,b = 88.63,c= 86.15 Å, α = 90.00, β = 114.30, γ = 90.00°. A molecular-replacement solution was obtained using the crystal structure ofMycobacterium smegmatisarylesterase as a template and structure refinement of Est24 is in progress.

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis ofAspergillus terreusendo-β-1,4-glucanase from glycoside hydrolase family 12

2014 ◽  
Vol 70 (2) ◽  
pp. 267-270 ◽  
Author(s):  
Fernando Segato ◽  
Gabriela L. Berto ◽  
Evandro Ares de Araújo ◽  
João Renato Muniz ◽  
Igor Polikarpov
Keyword(s):  
Glycoside Hydrolase ◽  
Aspergillus Terreus ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Hydrolase Family

Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such asAspergillus terreusare effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). TheA. terreusGH12 endoglucanase was cloned and overexpressed inA. nidulans, purified and crystallized. A single crystal was obtained from a solution consisting of 2 Mammonium sulfate, 5%(v/v) 2-propanol. X-ray diffraction data were collected to a resolution of 1.85 Å using synchrotron radiation and a preliminary molecular-replacement solution was obtained in the trigonal space groupP3221. The unit-cell parameters werea=b= 103.24,c= 48.96 Å.

Crystallization and preliminary X-ray diffraction studies of piratoxin III, a D-49 phospholipase A2 from the venom of Bothrops pirajai

1999 ◽  
Vol 55 (6) ◽  
pp. 1229-1230 ◽  
Author(s):  
Lee Wen-Hwa ◽  
Marcos H. Toyama ◽  
Andreimar M. Soares ◽  
José R. Giglio ◽  
Sérgio Marangoni ◽  
...  
Keyword(s):  
Search Model ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Diffusion Technique ◽  
Vapour Diffusion ◽  
Replacement Solution ◽  
Bothrops Pirajai

Piratoxin III (PrTX-III) is a phospholipase A2 (PLA2, E.C. 3.1.1.4, phosphatide sn-2 acylhydrolase) isolated from Bothrops pirajai. Crystals of PrTX-III were obtained using the vapour-diffusion technique and X-ray diffraction data have been collected to 2.7 Å resolution. The enzyme was crystallized in the space group C2 with unit-cell parameters a = 60.88, b = 100.75, c = 48.19 Å, β = 123.89°. A molecular-replacement solution of the structure has been found using bothropstoxin I from the venom of B. jararacussu as a search model.

scholarly journals Crystallization and preliminary X-ray analysis of a novel type of lipolytic hydrolase fromBacillus licheniformis

2014 ◽  
Vol 70 (4) ◽  
pp. 473-475
Author(s):  
Hansol Ju ◽  
Ramesh Pandian ◽  
Kyungmin Kim ◽  
Kyeong Kyu Kim ◽  
T. Doohun Kim
Keyword(s):  
Structure Refinement ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Peg 4000 ◽  
Replacement Solution ◽  
Increasing Demand ◽  
Identification And Characterization

With increasing demand in biotechnological applications, the identification and characterization of novel lipolytic enzymes are of great importance. The crystallization and preliminary X-ray crystallographic study of a novel type of hydrolase fromBacillus licheniformis(BL28) are described here. Recombinant BL28 protein containing a C-terminal His tag was overproduced inEscherichia coliand purified to homogeneity. BL28 was crystallized using 0.2 Mammonium acetate, 0.1 Msodium citrate tribasic dihydrate pH 5.6, 30%(w/v) PEG 4000 as a crystallizing solution. X-ray diffraction data were collected to a resolution of 1.67 Å with anRmergeof 5.8%. The BL28 crystals belonged to the tetragonal space groupP41212, with unit-cell parametersa=b= 57.89,c= 167.25 Å. A molecular-replacement solution was obtained and structure refinement of BL28 is in progress.

Crystallization and preliminary X-ray diffraction studies of human catalase

1999 ◽  
Vol 55 (9) ◽  
pp. 1614-1615 ◽  
Author(s):  
R. A. P. Nagem ◽  
E. A. L. Martins ◽  
V. M. Gonçalves ◽  
R. Aparício ◽  
I. Polikarpov
Keyword(s):  
Search Model ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Beef Liver ◽  
X Ray ◽  
Diffusion Technique ◽  
Cell Dimensions ◽  
Synchrotron Radiation Diffraction ◽  
Replacement Solution ◽  
Unit Cell Dimensions

The enzyme catalase (H2O2–H2O2 oxidoreductase; E.C. 11.1.6) was purified from haemolysate of human placenta and crystallized using the vapour-diffusion technique. Synchrotron-radiation diffraction data have been collected to 1.76 Å resolution. The enzyme crystallized in the space group P212121, with unit-cell dimensions a = 83.6, b = 139.4, c = 227.5 Å. A molecular-replacement solution of the structure has been obtained using beef liver catalase (PDB code 4blc) as a search model.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) fromCoffea canephorainvolved in chlorogenic acid biosynthesis

2012 ◽  
Vol 68 (7) ◽  
pp. 824-828 ◽  
Author(s):  
Laura A. Lallemand ◽  
James G. McCarthy ◽  
Sean McSweeney ◽  
Andrew A. McCarthy
Keyword(s):  
Structural Data ◽  
Coffea Canephora ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Key Enzyme ◽  
Substrate Specifities ◽  
Drop Technique ◽  
Better Than

Chlorogenic acids (CGAs) are a group of soluble phenolic compounds that are produced by a variety of plants, includingCoffea canephora(robusta coffee). The last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (HQT), but it can also be catalysed by the more widely distributed hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT). Here, the cloning and overexpression of HCT fromC. canephorainEscherichia colias well as its purification and crystallization are presented. Crystals were obtained by the sitting-drop technique at 293 K and X-ray diffraction data were collected on the microfocus beamline ID23-2 at the ESRF. The HCT crystals diffracted to better than 3.0 Å resolution, belonged to space groupP42212 with unit-cell parametersa=b= 116.1,c= 158.9 Å and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement and is currently under refinement. Such structural data are needed to decipher the molecular basis of the substrate specifities of this key enzyme, which belongs to the large plant acyl-CoA-dependent BAHD acyltransferase superfamily.

scholarly journals MSMEG_6292, a Mycobacterium smegmatis RNA polymerase secondary channel-binding protein: purification, crystallization and X-ray diffraction analysis

2018 ◽  
Vol 74 (9) ◽  
pp. 543-548
Author(s):  
Abyson Joseph ◽  
Valakunja Nagaraja ◽  
Ramanathan Natesh
Keyword(s):  
Rna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Transcriptional Activity ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Secondary Channel ◽  
X Ray ◽  
Cell Parameters ◽  
Crystallization Method ◽  
Better Than

The transcriptional activity of RNA polymerase (RNAP) is controlled by a diverse set of regulatory factors. A subset of these regulators modulate the activity of RNAP through its secondary channel. Gre factors reactivate stalled elongation complexes by enhancing the intrinsic cleavage activity of RNAP. In the present study, the protein MSMEG_6292, a Gre-factor homologue from Mycobacterium smegmatis, was expressed heterologously in Escherichia coli and purified using standard chromatographic techniques. The hanging-drop vapour-diffusion crystallization method yielded diffraction-quality crystals. The crystals belonged to the trigonal space group P3121 (or its enantiomorph P3221), with unit-cell parameters a = b = 83.15, c = 107.07 Å, α = β = 90, γ = 120°. The crystals diffracted to better than 3.0 Å resolution. Molecular-replacement attempts did not yield any phasing models; hence, platinum derivatization was carried out with K2PtCl4 and derivative data were collected to 3.4 Å resolution.

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