scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogenStaphylococcus aureus

2015 ◽  
Vol 71 (4) ◽  
pp. 477-482 ◽  
Author(s):  
Ana Lúcia Rosário ◽  
Filipa V. Sena ◽  
Ana P. Batista ◽  
Tânia F. Oliveira ◽  
Diogo Athayde ◽  
...  
Keyword(s):  
Drug Targets ◽  
Model Building ◽  
Type Ii ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
Cell Parameters ◽  
Nadh:Quinone Oxidoreductase ◽  
Wide Range ◽  
Replacement Solution ◽  
Causative Agents

In recent years, type II NADH dehydrogenases (NDH-IIs) have emerged as potential drug targets for a wide range of human disease causative agents. In this work, the NDH-II enzyme from the Gram-positive human pathogenStaphylococcus aureuswas recombinantly expressed inEscherichia coli, purified, crystallized and a crystallographic data set was collected at a wavelength of 0.873 Å. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 81.8,b= 86.0,c= 269.9 Å, contained four monomers per asymmetric unit and diffracted to a resolution of 3.32 Å. A molecular-replacement solution was obtained and model building and refinement are currently under way.

scholarly journals Preliminary crystallographic analysis of a double mutant of the acetyl xylo-oligosaccharide esterase Axe2 in its dimeric form

2014 ◽  
Vol 70 (4) ◽  
pp. 476-481 ◽  
Author(s):  
Shifra Lansky ◽  
Onit Alalouf ◽  
Rachel Salama ◽  
Hay Dvir ◽  
Yuval Shoham ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Plant Biomass ◽  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
Cell Parameters ◽  
Wide Range ◽  
Dimeric Form

Xylans are polymeric sugars constituting a significant part of the plant cell wall. They are usually substituted with acetyl side groups attached at positions 2 or 3 of the xylose backbone units. Acetylxylan esterases are part of the hemicellulolytic system of many microorganisms which utilize plant biomass for growth. These enzymes hydrolyze the ester linkages of the xylan acetyl groups and thus improve the accessibility of main-chain-hydrolyzing enzymes and their ability to break down the sugar backbone units. The acetylxylan esterases are therefore critically important for those microorganisms and as such could be used for a wide range of biotechnological applications. The structure of an acetylxylan esterase (Axe2) isolated from the thermophilic bacteriumGeobacillus stearothermophilusT6 has been determined, and it has been demonstrated that the wild-type enzyme is present as a unique torus-shaped octamer in the crystal and in solution. In order to understand the functional origin of this unique oligomeric structure, a series of rational noncatalytic, site-specific mutations have been made on Axe2. Some of these mutations led to a different dimeric form of the protein, which showed a significant reduction in catalytic activity. One of these double mutants, Axe2-Y184F-W190P, has recently been overexpressed, purified and crystallized. The best crystals obtained belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 71.1,b= 106.0,c= 378.6 Å. A full diffraction data set to 2.3 Å resolution has been collected from a flash-cooled crystal of this type at 100 K using synchrotron radiation. This data set is currently being used for the three-dimensional structure analysis of the Axe2-Y184F-W190P mutant in its dimeric form.

scholarly journals Recombinant production, purification and crystallization of theToxoplasma gondiicoronin WD40 domain

2014 ◽  
Vol 70 (4) ◽  
pp. 517-521 ◽  
Author(s):  
Juha Pekka Kallio ◽  
Inari Kursula
Keyword(s):  
Coiled Coil ◽  
Gliding Motility ◽  
Actin Binding ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
Cell Parameters ◽  
Recombinant Production ◽  
Small Set ◽  
Wd40 Domain ◽  
Conserved Region

Toxoplasma gondiiis one of the most widely spread parasitic organisms in the world. Together with other apicomplexan parasites, it utilizes a special actin–myosin motor for its cellular movement, called gliding motility. This actin-based process is regulated by a small set of actin-binding proteins, which in Apicomplexa comprises only 10–15 proteins, compared with >150 in higher eukaryotes. Coronin is a highly conserved regulator of the actin cytoskeleton, but its functions, especially in parasites, have remained enigmatic. Coronins consist of an N-terminal actin-binding β-propeller WD40 domain, followed by a conserved region, and a C-terminal coiled-coil domain implicated in oligomerization. Here, the WD40 domain and the conserved region of coronin fromT. gondiiwere produced recombinantly and crystallized. A single-wavelength diffraction data set was collected to a resolution of 1.65 Å. The crystal belonged to the orthorhombic space groupC2221, with unit-cell parametersa= 55.13,b= 82.51,c= 156.98 Å.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of the homing endonuclease I-CvuI fromChlorella vulgarisin complex with its target DNA

2014 ◽  
Vol 70 (2) ◽  
pp. 256-259 ◽  
Author(s):  
Pilar Redondo ◽  
Nekane Merino ◽  
Maider Villate ◽  
Francisco J. Blanco ◽  
Guillermo Montoya ◽  
...  
Keyword(s):  
Diffraction Analysis ◽  
Homing Endonuclease ◽  
Homing Endonucleases ◽  
Resolution Limit ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
Strand Breaks ◽  
X Ray ◽  
Cell Parameters ◽  
Wide Range

Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of DNA. The engineering of these enzymes provides novel instruments for genome modification in a wide range of fields, including gene targeting, by inducing specific double-strand breaks. I-CvuI is a homing endonuclease from the green algaChlorella vulgaris. This enzyme was purified after overexpression inEscherichia coli. Crystallization experiments of I-CvuI in complex with its DNA target in the presence of Mg2+yielded crystals suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 62.83,b= 83.56,c= 94.40 Å. The self-rotation function and the Matthews coefficient suggested the presence of one protein–DNA complex per asymmetric unit. The crystals diffracted to a resolution limit of 1.9 Å using synchrotron radiation.

scholarly journals Structure of the I-SceI nuclease complexed with its dsDNA target and three catalytic metal ions

2016 ◽  
Vol 72 (6) ◽  
pp. 473-479 ◽  
Author(s):  
Jesús Prieto ◽  
Pilar Redondo ◽  
Nekane Merino ◽  
Maider Villate ◽  
Guillermo Montoya ◽  
...  
Keyword(s):  
Metal Ion ◽  
Catalytic Mechanism ◽  
Homing Endonuclease ◽  
Homing Endonucleases ◽  
Resolution Limit ◽  
Orthorhombic Space Group ◽  
Cell Parameters ◽  
Anomalous Data ◽  
Wide Range ◽  
Dna Strand

Homing endonucleases are highly specific DNA-cleaving enzymes that recognize and cleave long stretches of DNA. The engineering of these enzymes provides instruments for genome modification in a wide range of fields, including gene targeting. The homing endonuclease I-SceI from the yeastSaccharomyces cerevisiaehas been purified after overexpression inEscherichia coliand its crystal structure has been determined in complex with its target DNA. In order to evaluate the number of ions that are involved in the cleavage process, thus determining the catalytic mechanism, crystallization experiments were performed in the presence of Mn2+, yielding crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 80.11,b= 80.57,c= 130.87 Å, α = β = γ = 90°. The self-rotation function and the Matthews coefficient suggested the presence of two protein–DNA complexes in the asymmetric unit. The crystals diffracted to a resolution limit of 2.9 Å using synchrotron radiation. From the anomalous data, it was determined that three cations are involved in catalysis and it was confirmed that I-SceI follows a two-metal-ion DNA-strand cleavage mechanism.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of an endo-1,4-β-D-glucanase fromAspergillus aculeatusF-50

2015 ◽  
Vol 71 (4) ◽  
pp. 397-400 ◽  
Author(s):  
Yun Chen ◽  
Jian-Wen Huang ◽  
Chun-Chi Chen ◽  
Hui-Lin Lai ◽  
Jian Jin ◽  
...  
Keyword(s):  
Model Building ◽  
Structure Refinement ◽  
Diffusion Method ◽  
Cellulolytic Enzyme ◽  
Glycoside Hydrolase Family ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
Cell Parameters ◽  
Renewable Biomass ◽  
Glycosidic Bonds

Cellulose is the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in a wide variety of industries. Endo-1,4-β-D-glucanase (EC 3.2.1.4; endoglucanase), which can catalyze the random hydrolysis of β-1,4-glycosidic bonds to cleave cellulose into smaller fragments, is a key cellulolytic enzyme. An endoglucanase isolated fromAspergillus aculeatusF-50 (FI-CMCase) that was classified into glycoside hydrolase family 12 has been found to be effectively expressed in the industrial strainPichia pastoris. Here, recombinant FI-CMCase was crystallized. Crystals belonging to the orthorhombic space groupC2221, with unit-cell parametersa= 74.2,b= 75.1,c= 188.4 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.6 Å resolution. Initial phase determination by molecular replacement clearly shows that the crystal contains two protein molecules in the asymmetric unit. Further model building and structure refinement are in progress.

Crystallization and preliminary crystallographic study of HIP/PAP, a human C-lectin overexpressed in primary liver cancers

1999 ◽  
Vol 55 (8) ◽  
pp. 1487-1489 ◽  
Author(s):  
Chantal Abergel ◽  
Sabine Chenivesse ◽  
Marie-Georges Stinnakre ◽  
Sophie Guasco ◽  
Christian Bréchot ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Adhesion Protein ◽  
Cell Parameters ◽  
Crystallographic Study ◽  
Liver Cancers ◽  
Replacement Solution

Human HIP/PAP is an adhesion protein expressed in normal pancreatic and Paneth cells and overexpressed in hepatocellular carcinoma. HIP/PAP was crystallized using the Hampton Research Crystal Screen and SAmBA software to define the optimal crystallization protocol. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 30.73, b = 49.35, c = 92.15 Å and one molecule in the asymmetric unit. Flash-frozen crystals diffract to 1.78 Å resolution using synchrotron radiation. A molecular-replacement solution was obtained using the human Reg/lithostathine structure and the AMoRe software.

Crystallization of ClfA and ClfB fragments: the fibrinogen-binding surface proteins of Staphylococcus aureus

1999 ◽  
Vol 55 (2) ◽  
pp. 554-556 ◽  
Author(s):  
Champion C. S. Deivanayagam ◽  
Samuel Perkins ◽  
Sita Danthuluri ◽  
Rick T. Owens ◽  
Todd Bice ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Unit Cell ◽  
Surface Proteins ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
Space Group ◽  
Cell Parameters ◽  
Fibrinogen Binding

Recombinant constructs encoding the fibrinogen-binding domains of ClfA and ClfB from Staphylococcus aureus have been crystallized. ClfA was crystallized in the orthorhombic space group P212121 with unit-cell parameters a = 39.58, b = 81.39 and c = 112.65 Å. A complete data set was recorded to 2.1 Å resolution and had a Vm of 2.3 Å3 Da−1 with 46.5% solvent, suggesting one molecule per asymmetric unit. Co-crystals of ClfA with the 17 amino-acid C-terminal peptide of fibrinogen γ-chain diffracted to 2.1 Å resolution and had unit-cell parameters a = 39.11, b = 81.39 and c = 109.51 Å in the space group P212121. ClfB was crystallized in the tetragonal space group P41212 or P43212 with unit-cell parameters a = 96.31, b = 96.31 and c = 84.13 Å and diffracted to 2.45 Å resolution. The estimated Vm of 2.6 Å3 Da−1 with 53% solvent indicated one molecule in the asymmetric unit.

scholarly journals Crystallization and preliminary crystallographic analysis of poly(3-hydroxybutyrate) depolymerase fromBacillus thuringiensis

2014 ◽  
Vol 70 (10) ◽  
pp. 1421-1423 ◽  
Author(s):  
Yung-Lin Wang ◽  
Yi-Ting Lin ◽  
Chia-Lin Chen ◽  
Gwo-Chyuan Shaw ◽  
Shwu-Huey Liaw
Keyword(s):  
Crystal Structure ◽  
Crystallographic Analysis ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Data Set ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Potential Applications ◽  
Polymer Biodegradation ◽  
Key Enzyme

Poly[(R)-3-hydroxybutyrate] (PHB) is a microbial biopolymer that has been commercialized as biodegradable plastics. The key enzyme for the degradation is PHB depolymerase (PhaZ). A new intracellular PhaZ fromBacillus thuringiensis(BtPhaZ) has been screened for potential applications in polymer biodegradation. Recombinant BtPhaZ was crystallized using 25% polyethylene glycol 3350, 0.2 Mammonium acetate, 0.1 Mbis-tris pH 6.5 at 288 K. The crystals belonged to space groupP1, with unit-cell parametersa= 42.97,b= 83.23,c= 85.50 Å, α = 73.45, β = 82.83, γ = 83.49°. An X-ray diffraction data set was collected to 1.42 Å resolution with anRmergeof 6.4%. Unexpectedly, a molecular-replacement solution was obtained using the crystal structure ofStreptomyces lividanschloroperoxidase as a template, which shares 24% sequence identity to BtPhaZ. This is the first crystal structure of an intracellular poly(3-hydroxybutyrate) depolymerase.

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis of the dihydroorotase domain of human CAD

2012 ◽  
Vol 68 (11) ◽  
pp. 1341-1345 ◽  
Author(s):  
Nada Lallous ◽  
Araceli Grande-García ◽  
Rafael Molina ◽  
Santiago Ramón-Maiques
Keyword(s):  
De Novo ◽  
Structural Information ◽  
Protein Molecule ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Small Crystals ◽  
Crystallization Experiments

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions ofde novopyrimidine biosynthesis: glutamine-dependentcarbamyl phosphate synthetase,aspartate transcarbamylase anddihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Å resolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space groupC2221, with unit-cell parametersa= 82.1,b= 159.3,c= 61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeastSaccharomyces cerevisiae

2015 ◽  
Vol 71 (2) ◽  
pp. 243-246 ◽  
Author(s):  
Srinivasan Rengachari ◽  
Philipp Aschauer ◽  
Christian Sturm ◽  
Monika Oberer
Keyword(s):  
Crystal Form ◽  
Size Exclusion ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Monoglyceride Lipase ◽  
Exclusion Chromatography

The protein Yju3p is the orthologue of monoglyceride lipases in the yeastSaccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space groupP212121), with unit-cell parametersa= 77.2,b= 108.6,c= 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

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