scholarly journals Expression, purification and preliminary crystallographic analysis ofMycobacterium tuberculosisCysQ, a phosphatase involved in sulfur metabolism

2014 ◽  
Vol 70 (6) ◽  
pp. 750-753 ◽  
Author(s):  
Anna I. Erickson ◽  
Reta D. Sarsam ◽  
Andrew J. Fisher
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sulfur Metabolism ◽  
Crystallographic Analysis ◽  
Transfer Reactions ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

CysQ is part of the sulfur-activation pathway that dephosphorylates 3′-phosphoadenosine 5′-monophosphate (PAP) to regenerate adenosine 5′-monophosphate (AMP) and free phosphate. PAP is the product of sulfate-transfer reactions from sulfotransferases that use the universal sulfate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS). In some organisms PAP is also the product of PAPS reductases that reduce sulfate from PAPS to sulfite. CysQ fromMycobacterium tuberculosis, which plays an important role in the biosynthesis of sulfated glycoconjugates, was successfully purified and crystallized in 24% PEG 1500, 20% glycerol. X-ray diffraction data were collected to 1.7 Å resolution using a synchrotron-radiation source. Crystals grew in the orthorhombic space groupP212121, with unit-cell parametersa= 40.3,b= 57.9,c= 101.7 Å and with one monomer per asymmetric unit.

scholarly journals Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase–RNA complex

2013 ◽  
Vol 69 (4) ◽  
pp. 421-424 ◽  
Author(s):  
Peter-Thomas Naumann ◽  
Charles T. Lauhon ◽  
Ralf Ficner
Keyword(s):  
Diffraction Data ◽  
Thermotoga Maritima ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Dependent Modification ◽  
Rna Complex

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI fromThermotoga maritimawas cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 102.9,b= 112.8,c= 132.8 Å.

scholarly journals Overproduction, crystallization and preliminary X-ray crystallographic analysis ofEscherichia colitRNAN6-threonylcarbamoyladenosine dehydratase

2014 ◽  
Vol 70 (11) ◽  
pp. 1517-1520 ◽  
Author(s):  
Sunmin Kim ◽  
Keon Young Kim ◽  
Jeong Kuk Park ◽  
Byung Il Lee ◽  
Yun-Gon Kim ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
X Rays ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Protein Mass ◽  
Crystal Volume

Escherichia colitRNAN6-threonylcarbamoyladenosine dehydratase (TcdA), previously called CsdL or YgdL, was overproduced and purified fromE. coliand crystallized using polyethylene glycol 3350 as a crystallizing agent. X-ray diffraction data were collected to 2.70 Å resolution under cryoconditions using synchrotron X-rays. The crystals belonged to space groupP21, with unit-cell parametersa= 65.4,b= 96.8,c= 83.3 Å, β = 111.7°. According to the Matthews coefficient, the asymmetric unit may contain up to four subunits of the monomeric protein, with a crystal volume per protein mass (VM) of 2.12 Å3 Da−1and 42.1% solvent content.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) fromBacillus cereus

2014 ◽  
Vol 70 (6) ◽  
pp. 777-780 ◽  
Author(s):  
Silje Skråmo ◽  
Hans-Petter Hersleth ◽  
Marta Hammerstad ◽  
K. Kristoffer Andersson ◽  
Åsmund K. Røhr
Keyword(s):  
Electron Transfer ◽  
Thioredoxin Reductase ◽  
Free Form ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) are key enzymes involved in catalysing electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. InBacillus cereusthere are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallized in its NADPH/NADP+-free form. Diffraction data have been collected to 2.5 Å resolution from crystals belonging to the orthorhombic space groupP21212, with unit-cell parametersa= 57.2,b= 164.3,c= 95.0 Å, containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs.

Crystallization and preliminary X-ray diffraction analysis of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

1999 ◽  
Vol 55 (6) ◽  
pp. 1231-1233 ◽  
Author(s):  
Ivana Smatanová ◽  
Yuji Nagata ◽  
L. Anders Svensson ◽  
Masamichi Takagi ◽  
Jaromír Marek
Keyword(s):  
Diffusion Method ◽  
Sphingomonas Paucimobilis ◽  
Asymmetric Unit ◽  
Haloalkane Dehalogenase ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18–20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris–HCl pH 8.9. The crystals diffract to at least 1.60 Å using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 Å. The asymmetric unit contains one molecule of the enzyme.

scholarly journals Expression, crystallization and preliminary X-ray crystallographic analysis ofD-alanine-D-alanine ligase from OXA-23-producingAcinetobacter baumanniiK0420859

2014 ◽  
Vol 70 (4) ◽  
pp. 505-508 ◽  
Author(s):  
Kim-Hung Huynh ◽  
Huyen-Thi Tran ◽  
Tan-Viet Pham ◽  
Ho-Phuong-Thuy Ngo ◽  
Sun-Shin Cha ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Antibacterial Drug ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Novel Target ◽  
Tract Infections

Acinetobacter baumanniicauses bacteraemia, pneumonia, other respiratory-tract and urinary-tract infections in humans. OXA-23 carbapenemase-producingA. baumanniiK0420859 (A. baumanniiOXA-23) is resistant to carbapenem, a common antibacterial drug. To develop an efficient and novel antibacterial drug againstA. baumanniiOXA-23, D-alanine-D-alanine ligase, which is essential in bacterial cell-wall synthesis, is of interest. Here, the D-alanine-D-alanine ligase (AbDdl) gene fromA. baumanniiOXA-23 was cloned and expressed, and theAbDdl protein was purified and crystallized; this enzyme can be used as a novel target for an antibacterial drug againstA. baumanniiOXA-23. TheAbDdl crystal diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 113.4,b= 116.7,c= 176.5 Å, a correspondingVMof 2.8 Å3 Da−1and a solvent content of 56.3%, and six protomers in the asymmetric unit.

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 Expression, purification and preliminary crystallographic analysis of a haem-utilizing protein, HutX, fromVibrio cholerae

2015 ◽  
Vol 71 (2) ◽  
pp. 141-144 ◽  
Author(s):  
Tiantian Su ◽  
Kaikai Chi ◽  
Kang Wang ◽  
Liming Guo ◽  
Yan Huang
Keyword(s):  
Iron Acquisition ◽  
Crystallographic Analysis ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Important Method ◽  
X Ray ◽  
Cell Parameters ◽  
Protein Molecules ◽  
Open Question

Vibrio cholerae, the causative agent of cholera, has developed a variety of mechanisms to obtain the limited-availability iron from human hosts. One important method for iron acquisition is through haem-uptake systems. Although the transport of haem has been widely studied, the fate of haem once it enters the cytoplasm remains an open question. Here, preliminary X-ray crystallographic analysis was performed on HutX, a member of the conserved haem-utilization operon fromV. choleraestrain N16961. The crystals of HutX were found to belong to the orthorhombic space groupC2221, with unit-cell parametersa= 50.1,b= 169.0,c= 81.8 Å. There are two protein molecules in the asymmetric unit, with a corresponding Matthews coefficientVMof 2.06 Å3 Da−1and a solvent content of 40.28%.

scholarly journals Crystallographic analysis of RsmA, a ribosomal RNA small subunit methyltransferase A fromStaphylococcus aureus

2015 ◽  
Vol 71 (8) ◽  
pp. 1063-1066 ◽  
Author(s):  
Yang Liu ◽  
Yuwei Zhu ◽  
Maikun Teng ◽  
Xu Li
Keyword(s):  
Staphylococcus Aureus ◽  
Ribosomal Rna ◽  
Small Subunit ◽  
Crystallographic Analysis ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Rotation Function

RsmA, a ribosomal RNA small subunit methyltransferase fromStaphylococcus aureus, catalyzes theN6methylation of adenine in 16S rRNA. In this study, RsmA fromStaphylococcus aureuswas cloned, expressed, purified and crystallized. The crystal belonged to space groupC2, with unit-cell parametersa= 84.38,b= 157.76,c= 96.50 Å, β = 95.04°. X-ray diffraction data were collected to a resolution of 3.2 Å. The self-rotation function and the Matthews coefficient suggested the presence of two molecules in the asymmetric unit.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of ribosome assembly factors: the Rpf2–Rrs1 complex

2014 ◽  
Vol 70 (12) ◽  
pp. 1649-1652 ◽  
Author(s):  
Nozomi Asano ◽  
Akiyoshi Nakamura ◽  
Keisuke Komoda ◽  
Koji Kato ◽  
Isao Tanaka ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Crystallographic Analysis ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Essential Proteins ◽  
Unit Structure

Rpf2 and Rrs1 are essential proteins for ribosome biogenesis. These proteins form a complex (the Rpf2-subcomplex) with 5S rRNA and two ribosomal proteins (L5 and L11). This complex is recruited to the ribosome precursor (the 90S pre-ribosome). This recruitment is necessary for the maturation of 25S rRNA. Genetic depletion of Rpf2 and Rrs1 results in accumulation of the 25S rRNA precursor. In this study, Rpf2 and Rrs1 fromAspergillus nidulanswere co-overexpressed inEscherichia coli, purified and crystallized. Subsequent analysis revealed that these crystals contained the central core region of the complex consisting of both N-terminal domains. X-ray diffraction data were collected to 2.35 Å resolution. Preliminary analysis revealed that the crystals belonged to space groupP212121, with unit-cell parametersa= 54.1,b= 123.3,c = 133.8 Å. There are two complexes in the asymmetric unit. Structure determination using selenomethionine-labelled protein is in progress.

scholarly journals Ectodomain of plasmodesmata-localized protein 5 inArabidopsis: expression, purification, crystallization and crystallographic analysis

2017 ◽  
Vol 73 (9) ◽  
pp. 532-535 ◽  
Author(s):  
Xiaocui Wang ◽  
Peiyan Zhu ◽  
Shanshan Qu ◽  
Jie Zhao ◽  
Prashant K. Singh ◽  
...  
Keyword(s):  
Expression System ◽  
Cell Movement ◽  
Crystallographic Analysis ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Crystal Content

Plasmodesmata-localized protein 5 (PDLP5) is a cysteine-rich receptor-like protein which is localized on the plasmodesmata ofArabidopsis thaliana. Overexpression of PDLP5 can reduce the permeability of the plasmodesmata and further affect the cell-to-cell movement of viruses and macromolecules in plants. The ectodomain of PDLP5 contains two DUF26 domains; however, the function of these domains is still unknown. Here, the ectodomain of PDLP5 fromArabidopsiswas cloned and overexpressed using an insect expression system and was then purified and crystallized. X-ray diffraction data were collected to 1.90 Å resolution and were indexed in space groupP1, with unit-cell parametersa= 41.9,b= 48.1,c = 62.2 Å, α = 97.3, β = 103.1, γ = 99.7°. Analysis of the crystal content indicated that there are two molecules in the asymmetric unit, with a Matthews coefficient of 2.51 Å3 Da−1and a solvent content of 50.97%.

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