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Preliminary Crystallographic Analysis (preliminary + crystallographic_analysis)

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Selected Abstracts

Crystallization and preliminary crystallographic analysis of a family 45 endoglucanase from the thermophilic fungus Melanocarpus albomyces

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002
Mika Hirvonen
20K-cellulase, an endoglucanase produced by the thermophilic fungus Melanocarpus albomyces, has been crystallized. It belongs to the family 45 of glycoside hydrolases and has sequence homology with Humicola insolens endoglucanase V (EGV). However, in contrast to EGV, it does not harbour a cellulose-binding module. Optimization of the crystallization conditions using a PEG/ion combination and microseeding techniques was employed to improve the quality and the size of the crystals. A complete data set to 2.2,Å resolution was collected using synchrotron radiation. Preliminary crystallographic analysis showed that the crystals belong to the tetragonal space group P41212/P43212, with unit-cell parameters a = 47.3, b = 47.3, c = 177.3,Å and one molecule per asymmetric unit. [source]

Preliminary crystallographic analysis of the Escherichia coli antitoxin MqsA (YgiT/b3021) in complex with mqsRA promoter DNA

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010
Breann L. Brown
The Escherichia coli proteins MqsR and MqsA comprise a novel toxin,antitoxin (TA) system. MqsA, the antitoxin, defines a new family of antitoxins because unlike other antitoxins MqsA is structured throughout its entire sequence, binds zinc and coordinates DNA via its C-terminal and not its N-terminal domain. In order to understand how bacterial antitoxins, and MqsA in particular, regulate transcription, the MqsA protein was cocrystallized with a 26-mer duplex DNA corresponding to the palindromic region of the mqsRA promoter. The merohedrally twinned crystal belonged to space group P41, with unit-cell parameters a = 60.99, b = 60.99, c = 148.60,Å. A complete data set was collected to a resolution of 2.1,Å. The solvent content of the crystal was consistent with the presence of two MqsA molecules bound to the duplex DNA in the asymmetric unit. [source]

Preliminary crystallographic analysis of the N-terminal domain of FILIA, a protein essential for embryogenesis

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010
Juke Wang
FILIA is a component of the subcortical maternal complex that is essential for early stage embryogenesis. Its 6×His-tagged N-terminal domain was expressed in Escherichia coli and purified to homogeneity. Two types of crystals formed under different crystallization conditions during screening. Orthorhombic crystals appeared in a solution containing 1.4,M ammonium sulfate, 0.1,M Tris pH 8.2 and 12% glycerol, while tetragonal crystals were obtained using 15% PEG 4000 mixed with 0.1,M HEPES pH 7.5 and 15% 2-propanol. High-quality diffraction data were collected from the two crystal forms to resolutions of 1.8 and 2.2,Å, respectively, using synchrotron radiation. The Matthews coefficients indicated that the P212121 and P41212 crystals contained two molecules and one molecule per asymmetric unit, respectively. A selenomethionine-substituted sample failed to crystallize under the native conditions, but another orthorhombic crystal form was obtained under different conditions and anomalous diffraction data were collected. [source]

Crystallization and preliminary X-ray analysis of NADH:rubredoxin oxidoreductase from Clostridium acetobutylicum

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010
Koji Nishikawa
NADH:rubredoxin oxidoreductase (NROR), an O2 -inducible protein, is a versatile electron donor for scavengers of O2 and reactive oxygen species (ROS) in Clostridium acetobutylicum. Recombinant NROR was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the sitting-drop vapour-diffusion method at 293,K. Preliminary crystallographic analysis revealed that the crystals belonged to space group P4122 or P4322, with unit-cell parameters a = b = 98.6, c = 88.3,Å, and diffracted to 2.1,Å resolution. Assuming that the crystals contained one molecule per asymmetric unit, the Matthews coefficient was calculated to be 2.7,Å3,Da,1 and the solvent content to be 54.1%. [source]

Preliminary crystallographic analysis of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-, receptor promoter DNA

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009
Vinod B. Agarkar
Ets proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. Like other Ets-family members, Elf3 functions as a sequence-specific DNA-binding transcriptional factor. A mouse Elf3 C-terminal fragment (amino-acid residues 269,371) containing the DNA-binding domain has been crystallized in complex with mouse type II TGF-, receptor promoter (T,R-II) DNA. The crystals belonged to space group P212121, with unit-cell parameters a = 42.66, b = 52, c = 99.78,Å, and diffracted to a resolution of 2.2,Å. [source]

Preliminary X-ray crystallographic analysis of sulfide:quinone oxidoreductase from Acidithiobacillus ferrooxidans

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009
Yanfei Zhang
The gene product of open reading frame AFE_1293 from Acidithiobacillus ferrooxidans ATCC 23270 is annotated as encoding a sulfide:quinone oxidoreductase, an enzyme that catalyses electron transfer from sulfide to quinone. Following overexpression in Escherichia coli, the enzyme was purified and crystallized using the hanging-drop vapour-diffusion method. The native crystals belonged to the tetragonal space group P42212, with unit-cell parameters a = b = 131.7, c = 208.8,Å, and diffracted to 2.3,Å resolution. Preliminary crystallographic analysis indicated the presence of a dimer in the asymmetric unit, with an extreme value of the Matthews coefficient (VM) of 4.53,Å3,Da,1 and a solvent content of 72.9%. [source]

Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009
Dipak N. Patil
A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS,PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21,kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 37.2, b = 77.1, c = 129.1,Å. Diffraction data were collected to a resolution of 2.7,Å. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%. [source]

Cloning, purification, crystallization and preliminary crystallographic analysis of a ribokinase from Staphylococcus aureus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009
Lin Wang
The gene SA239 from Staphylococcus aureus encodes a ribokinase that catalyzes the phosphorylation of d -ribose to produce ribose-5-phosphate. Sa239 was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.9,Å resolution and belonged to space group P6122 or P6522, with unit-cell parameters a = b = 91.8, c = 160.7,Å. Preliminary crystallographic analysis revealed that the Matthews coefficient VM was 3.01,Å3,Da,1, indicating the presence of one molecule in the asymmetric unit. [source]

Purification, crystallization and preliminary X-ray analysis of an aminoacylhistidine dipeptidase (PepD) from Vibrio alginolyticus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009
Chin-Yuan Chang
The aminoacylhistidine dipeptidase (PepD) protein encoded by Vibrio alginolyticus pepD was successfully overexpressed and characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The purified enzyme contained two zinc ions per monomer. The recombinant dipeptidase enzyme, which was identified as a homodimer in solution, exhibited broad substrate specificity for Xaa-His dipeptides, with highest activity towards the His-His dipeptide. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Preliminary crystallographic analysis showed that the crystal belonged to space group P61 or P65, with unit-cell parameters a = b = 80.42, c = 303.11,Å. The crystal contained two molecules per asymmetric unit and the predicted solvent content was 53.4%. [source]

Crystallization, diffraction data collection and preliminary crystallographic analysis of DING protein from Pseudomonas fluorescens

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2007
Sebastien Moniot
PfluDING is a phosphate-binding protein expressed in Pseudomonas fluorescens. This protein is clearly distinct from the bacterial ABC transporter soluble phosphate-binding protein PstS and is more homologous to eukaryotic DING proteins. Interestingly, bacterial DING proteins have only been detected in certain Pseudomonas species. Although DING proteins seem to be ubiquitous in eukaryotes, they are systematically absent from eukaryotic genomic databases and thus are still quite mysterious and poorly characterized. PfluDING displays mitogenic activity towards human cells and binds various ligands such as inorganic phosphate, pyrophosphate, nucleotide triphosphates and cotinine. Here, the crystallization of PfluDING is reported in a monoclinic space group (P21), with typical unit-cell parameters a = 36.7, b = 123.7, c = 40.8,Å, , = 90, , = 116.7, , = 90°. Preliminary crystallographic analysis reveals good diffraction quality for these crystals and a 1.43,Å resolution data set has been collected. [source]

Preliminary crystallographic analysis of the Escherichia coli YeaZ protein using the anomalous signal of a gadolinium derivative

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2005
Richard Kahn
The Escherichia coli yeaZ gene encodes a 231-residue protein (Mr = 25,180) that belongs to a family of proteins that are conserved in various bacterial genomes. This protein of unknown function is predicted to be a hypothetical protease. The YeaZ protein was overexpressed in E. coli and crystallized at 298,K by the hanging-drop vapour-diffusion method. A MAD data set was collected using a gadolinium-derivative crystal that had been soaked with 0.1,M Gd-DOTMA. The data set contained data collected to a resolution of 2.7,Å at two wavelengths at the LIII absorption edge of gadolinium, while remote data were collected to a resolution of 2.28,Å. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 76.3, b = 97.6, c = 141.9,Å. Phasing using the MAD method confirmed there to be four monomers in the asymmetric unit related by two twofold axes as identified by the self-rotation function search. [source]

Purification, crystallization and preliminary X-ray analysis of Triatoma virus (TrV) from Triatoma infestans

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004
Gabriela S. Rozas-Dennis
Triatoma virus (TrV) is a viral pathogen of the blood-sucking reduviid bug Triatoma infestans, the most important vector of American human trypanosomiasis (Chagas' disease). TrV has been putatively classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work describes the purification of TrV particles from infected T. infestans and their crystallization and preliminary crystallographic analyses. Two different crystal forms, rhombohedral and orthorhombic, were obtained at room temperature by the hanging-drop vapour-diffusion technique using polyethylene glycol and polyethylene glycol monomethylether as precipitants. The rhombohedral crystals have unit-cell parameters a = b = 306.6, c = 788.4,Å (hexagonal setting), diffract to 3.2,Å resolution and contain one-third of the viral particle per asymmetric unit. The orthorhombic crystals have cell parameters a = 336, b = 351, c = 332,Å, diffract to about 2.5,Å resolution, and contain one-half of a virus particle in the asymmetric unit. A complete diffraction data set has been collected to 3.2,Å resolution, using synchrotron radiation, from a single rhombohedral crystal under cryogenic conditions. [source]

Crystallization and preliminary X-ray diffraction analysis of various enzyme,substrate complexes of isopropylmalate dehydrogenase from Thermus thermophilus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010
Angelo Merli
The Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt -IPMDH) enzyme catalyses the penultimate step of the leucine-biosynthesis pathway. It converts (2R,3S)-3-isopropylmalate to (2S)-2-isopropyl-3-oxosuccinate in the presence of divalent Mg2+ or Mn2+ and with the help of NAD+. In order to elucidate the detailed structural and functional mode of the enzymatic reaction, crystals of Tt -IPMDH were grown in the presence of various combinations of substrate and/or cofactors. Here, the crystallization, data collection and preliminary crystallographic analyses of six such complexes are reported. [source]

Crystallization and preliminary X-ray crystallographic analysis of two dimeric hyperthermostable thioredoxins isolated from Sulfolobus solfataricus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009
Alessia Ruggiero
The thioredoxin system of the archaeon Sulfolobus solfataricus involves a number of different proteins: two thioredoxin reductases (SsTrxRB2 and SsTrxRB3), two distinct thioredoxins (SsTrxA1 and SsTrxA2) and a disulfide oxidoreductase (SsPDO). Here, the crystallization and preliminary crystallographic analyses of SsTrxA1 and SsTrxA2, two dimeric proteins endowed with extraordinary thermal stability, are reported. In addition to the functional thioredoxin domain, both SsTrxA1 and SsTrxA2 present an extra N-terminal fragment of approximately 30 residues. Although crystallization trials have been conducted on both forms of the proteins, crystals that were suitable for X-ray crystallographic analyses have only been obtained for their truncated variants. The crystals of SsTrxA2 belonged to space group P2, with unit-cell parameters a = 28.27, b = 27.88, c = 62.06,Å, , = 92.34°, and diffracted to 1.83,Å resolution, whereas the crystals of SsTrxA1 belonged to space group P21, with unit-cell parameters a = 51.76, b = 75.09, c = 55.35,Å, , = 112.64°, and diffracted to 1.90,Å resolution. The structures of the two proteins have been solved by molecular replacement. [source]

Crystallization and preliminary crystallographic analysis of endonuclease VIII in its uncomplexed form

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004
Gali Golan
The Escherichia coli DNA repair enzyme endonuclease VIII (EndoVIII or Nei) excises oxidized pyrimidines from damaged DNA substrates. It overlaps in substrate specificity with endonuclease III and may serve as a back-up for this enzyme in E. coli. The three-dimensional structure of Nei covalently complexed with DNA has been recently determined, revealing the critical amino-acid residues required for DNA binding and catalytic activity. Based on this information, several site-specific mutants of the enzyme have been tested for activity against various substrates. Although the crystal structure of the DNA-bound enzyme has been fully determined, the important structure of the free enzyme has not previously been analyzed. In this report, the crystallization and preliminary crystallographic characterization of DNA-free Nei are described. Four different crystal habits are reported for wild-type Nei and two of its catalytic mutants. Despite being crystallized under different conditions, all habits belong to the same crystal form, with the same space group (I222) and a similar crystallographic unit cell (average parameters a = 57.7, b = 80.2, c = 169.7,Å). Two of these crystal habits, I and IV, appear to be suitable for full crystallographic analysis. Crystal habit I was obtained by vapour diffusion using PEG 8000, glycerol and calcium acetate. Crystal habit IV was obtained by a similar method using PEG 400 and magnesium chloride. Both crystals are mechanically strong and stable in the X-ray beam once frozen under cold nitrogen gas. A full diffraction data set has recently been collected from a wild-type Nei crystal of habit I (2.6,Å resolution, 85.2% completeness, Rmerge = 9.8%). Additional diffraction data were collected from an Nei-R252A crystal of habit IV (2.05,Å resolution, 99.9% completeness, Rmerge = 6.0%) and an Nei-E2A crystal of habit IV (2.25,Å resolution, 91.7% completeness, Rmerge = 6.2%). These diffraction data were collected at 95,100 K using a synchrotron X-ray source and a CCD area detector. All three data sets are currently being used to obtain crystallographic phasing via molecular-replacement techniques. [source]

Crystallization and preliminary crystallographic analysis of a novel haemolytic lectin from the mushroom Laetiporus sulphureus

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2004
José M. Mancheño
The novel haemolytic lectin from the parasitic mushroom Laetiporus sulphureus (LSL) is a homotetramer (,140,kDa) composed of subunits associated by non-covalent bonds. It exhibits haemagglutin­ation and haemolytic activities, both of which are inhibited by N -­acetyllactosamine. The structural similarity found between LSL and the bacterial pore-forming toxins mosquitocidal toxin (MTX2) from Bacillus sphaericus and ,-toxin from Clostridium septicum points to a mechanism of biological action involving the formation of pores in the target membranes. LSL has been crystallized using the hanging-drop vapour-diffusion method at 291,K. Diffraction-quality hexagonal crystals have unit-cell parameters a = b = 101.8, c = 193.9,Å and belong to space group P6322. A 2.7,Å native data set was collected with an Rmerge of 9.2%. [source]

Overproduction, crystallization and preliminary crystallographic analysis of a novel human DNA-­repair enzyme that recognizes oxidative DNA damage

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2004
Viswanath Bandaru
DNA glycosylases repair oxidative DNA damage caused by free radicals. Recently, NEIL1, a human homolog of Escherichia coli DNA glycosylase endonuclease VIII, has been identified and shown to exhibit broad substrate specificity for a variety of types of pyrimidine-base damage. An active C-terminal deletion construct of NEIL1 was overexpressed in E. coli and crystallized. The unliganded NEIL1 crystallizes in space group R3, with unit-cell parameters a = b = 132.2, c = 51.1,Å. Complete data sets were collected from native, selenomethionyl and iodinated NEIL1 to 2.1, 2.3 and 2.4,Å, respectively. [source]

Crystallization, microPIXE and preliminary crystallographic analysis of the complex between the third KH domain of hnRNP K and single-stranded DNA

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004
Paul H. Backe
hnRNP K is one of the major proteins found in hnRNP particles which are ribonucleoprotein complexes containing proteins and pre-mRNA. hnRNP K contains hnRNP K homology (KH) domains which bind both CT-rich single-stranded DNA (ssDNA) and CU-rich ssRNA. Co-crystallization of the third KH domain of human hnRNP,K with a 15-mer ssDNA gave rod-shaped crystals belonging to the trigonal space group P3121 (unit-cell parameters a = 54.0, c = 149.7,Å) and diffracting to 2.4,Å resolution. MicroPIXE (proton-induced X-ray emission) experiments showed that the crystals contained the complex and that the phosphorus to sulfur atomic ratio was consistent with the asymmetric unit containing three KH3 domains per 15-mer ssDNA. This was confirmed by structure solution by molecular replacement. [source]

Expression, purification, crystallization and preliminary crystallographic analysis of the diarrhoea-causing and virulence-determining region of rotaviral nonstructural protein NSP4

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004
Rotaviral nonstructural protein NSP
The region spanning the tetrameric coiled-coil domain and the interspecies-variable virulence-determining region of the cytoplasmic tail of rotaviral nonstructural protein NSP4 has been crystallized. The crystals belong to space group I222, with unit-cell parameters a = 30.70, b = 38.07, c = 181.62,Å, and contain two molecules in the asymmetric unit. Diffraction data have been collected utilizing a MAR imaging plate to a resolution of 2.2,Å. The tetramer is generated by the crystallographic dyad along the c axis. [source]

Expression, purification, crystallization and preliminary crystallographic analysis of phosphoserine aminotransferase from Bacillus alcalophilus

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003
Anatoly P. Dubnovitsky
Phosphoserine aminotransferase (PSAT; EC 2.6.1.52) from Bacillus alcalophilus, an obligatory alkalophile with optimum growth at pH 10.6, was overexpressed in Escherichia coli, purified and crystallized under two different conditions using the hanging-drop vapour-diffusion method. Crystals were obtained using trisodium citrate dihydrate or PEG 400 as a precipitating agent. Crystals grown in the presence of trisodium citrate belong to the orthorhombic space group C2221, with unit-cell parameters a = 105.6, b = 136.6, c = 152.0,Å, and those grown in the presence of PEG 400 belong to the orthorhombic space group P21212, with unit-cell parameters a = 143.7, b = 84.3, c = 67.4,Å. Complete data sets were collected to 1.7 and 1.6,Å resolution, respectively, at 100,K using synchrotron radiation. Analysis of the structure of B. alcalophilus PSAT may reveal structural features that contribute to enzyme adaptability at high pH values. [source]

Crystallization and preliminary crystallographic analysis of extracellular fragment X3 of YWK-II/APPH: a human sperm membrane protein related to the Alzheimer ,A4-amyloid precursor protein

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003
Wangjun Hu
Crystals of extracellular fragment X3 of a human sperm membrane protein YWK-II/APPH have been grown at 291,K using 8% PEG 4000 as precipitant by the vapour-diffusion method. The diffraction pattern of the crystal extends to 2.9,Å resolution at 100,K using Cu,K, radiation in-house. The crystals belong to space group P21, with unit-cell parameters a = 46.0, b = 43.7, c = 90.2,Å, , = , = 90.0, , = 106.6°. Furthermore, a selenomethionine (SeMet) derivative of the protein was overexpressed in the same expression system and was purified in a reducing environment. The derivative crystals were obtained under similar conditions. Subsequently, a single-wavelength data set was collected to 2.38,Å resolution from the derivative crystal at ESRF. The crystals belong to space group P21, with unit-cell parameters a = 46.2, b = 44.0, c = 88.3,Å, , = , = 90.0, , = 103.6°. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (VM) of 2.8,Å3,Da,1 and a solvent content of 56.4% by volume. [source]

Purification, N-terminal sequencing, partial characterization, crystallization and preliminary crystallographic analysis of two glycosylated serine proteinases from Agkistrodon acutus venom

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003
Zhongliang Zhu
AaV-SP-I and AaV-SP-II, two glycosylated serine proteinases from Agkistrodon acutus venom with fibrinogenolysis and esterolysis activities, have been purified to homogeneity by three-step ion-exchange chromatography. Estimated by SDS,PAGE, the molecular weights of AaV-SP-I and AaV-SP-II are about 32 and 31,kDa under reducing conditions and 26 and 25,kDa under non-reducing conditions, respectively. The first 24 N-terminal amino-acid residues are the same in both sequences and display a high homology with those of several snake-venom serine proteinases. However, the proteins possess obviously distinct carbohydrate contents. Using the conventional hanging-drop vapour-diffusion method, single crystals of both enzymes were grown that were suitable for X-ray diffraction analysis. The crystals of AaV-SP-I and AaV-SP-II belong to space groups P212121 and C2, respectively. In each case there is only one molecule in the asymmetric unit. [source]

Crystallization and preliminary crystallographic analysis of a partial extracellular fragment of a sperm membrane protein YWK-II/APPH related to the Alzheimer ,A4-amyloid precursor protein

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2003
Maojun Yang
Crystals of a partial extracellular fragment of a human sperm membrane protein YWK-II/APPH have been grown at 291,K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.8,Å resolution at 100,K using Cu,K, radiation. The crystals belong to space group P212121, with unit-cell parameters a = 46.009, b = 67.387, c = 149.241,Å, , = , = , = 90°. The presence of two molecules per asymmetric unit gives a crystal volume per protein mass (VM) of 3.51,Å3,Da,1 and a solvent content of 64.6% by volume. A full set of X-ray diffraction data were collected to 2.8,Å resolution from the native crystal. [source]

Isolation, purification and preliminary X-ray characterization of Cpn60-2 (65,kDa heat-shock protein) from Mycobacterium tuberculosis

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2002
Noam Adir
Cpn60-2 is a member of a unique family of putative molecular chaperones homologous to GroEL (Cpn60) but of unknown function and found only in Mycobacterium tuberculosis and closely related species. Cpn60-2 has mainly been studied for its strong immunogenity. Here, the purification, crystallization and preliminary crystallographic analysis of M. tuberculosis Cpn60-2 are reported. The crystals belong to space group P2, with unit-cell parameters a = 57, b = 115.5, c = 81.5,Å, , = 95.5°, and contain a dimer in the asymmetric unit. The crystals diffract to 4.0,Å using a Cu rotating-anode X-ray generator. [source]

Preliminary X-ray crystallographic analysis of a soluble form of MntC, a periplasmic manganese-binding component of an ABC-type Mn transporter from Synechocystis sp.

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2002
PCC 680
Manganese is recruited in microorganisms by way of ABC-type transporter systems. Here, the expression, purification and preliminary crystallographic analysis of a soluble form of the MntC solute-binding protein component of the MntABC manganese-import system from the cyanobacterium Synechococystis sp. PCC 6803 is reported. The protein (321 amino-acid residues) was expressed exclusively in inclusion bodies, which required unfolding and refolding in the presence of manganese prior to purification. The purified protein was crystallized in the presence of PEG and zinc. The crystals belong to space group P6222, with unit-cell parameters a = b = 128.1, c = 90.0,Å and a single molecule in the asymmetric unit. The crystals diffract to 2.6,Å under cryoconditions using synchrotron radiation. [source]

Crystallization and preliminary crystallographic analysis of a family 45 endoglucanase from the thermophilic fungus Melanocarpus albomyces

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002
Mika Hirvonen
20K-cellulase, an endoglucanase produced by the thermophilic fungus Melanocarpus albomyces, has been crystallized. It belongs to the family 45 of glycoside hydrolases and has sequence homology with Humicola insolens endoglucanase V (EGV). However, in contrast to EGV, it does not harbour a cellulose-binding module. Optimization of the crystallization conditions using a PEG/ion combination and microseeding techniques was employed to improve the quality and the size of the crystals. A complete data set to 2.2,Å resolution was collected using synchrotron radiation. Preliminary crystallographic analysis showed that the crystals belong to the tetragonal space group P41212/P43212, with unit-cell parameters a = 47.3, b = 47.3, c = 177.3,Å and one molecule per asymmetric unit. [source]

Crystallization and preliminary crystallographic analysis of the recombinant N-terminal domain of riboflavin synthase

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001
Winfried Meining
Riboflavin synthase catalyzes the final step in the biosynthesis of riboflavin. Animals and humans lack this enzyme, whereas many bacteria and certain yeasts are absolutely dependent on endogenous riboflavin synthesis. Riboflavin synthase is therefore an attractive target for chemotherapy. The N-terminal domain of riboflavin synthase forms a dimer in solution and is capable of strongly binding riboflavin. It can serve as a model for the binding site of the native enzyme. Structural information obtained from this domain at high resolution will be helpful in the determination of the binding mode of riboflavin and thus for the development of antimicrobial drugs. Here, the crystallization and preliminary crystallographic analysis of the N-­terminal domain of riboflavin synthase are reported. The crystals belong to the space group C2221, with unit-cell parameters a = 50.3, b = 104.7, c = 85.3,Å, , = , = , = 90°, and diffract to 2.6,Å resolution. [source]

Crystallization and preliminary crystallographic analysis of Thermus thermophilus leucyl-tRNA synthetase and its complexes with leucine and a non-hydrolysable leucyl-adenylate analogue

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2000
Anna Yaremchuk
Leucyl-tRNA synthetase from Thermus thermophilus (LeuRSTT) is the first LeuRS to be crystallized. Two crystal forms of the native enzyme have been obtained using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant. Crystals of the first form belong to space group I422 and have unit-cell parameters a = b = 312.4, c = 100.4,Å. They diffract anisotropically to 3.5,Å resolution in the c -axis direction and to only 6,Å resolution in the perpendicular direction. Crystals of the second form, which can be obtained native or with leucine or a leucyl-adenylate analogue bound, belong to space group C2221 and have unit-cell parameters a = 102.4, b = 154.1, c = 174.3,Å. They diffract to 1.9,Å resolution and contain one monomer in the asymmetric unit. Selenomethionated LeuRSTT has been produced and crystals of the second form suitable for MAD analysis have been grown. [source]

Crystallization and preliminary crystallographic analysis of N -acetylglucosamine 6-phosphate deacetylase from Escherichia coli

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2000
F. M. Ferreira
N -Acetylglucosamine 6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme from Escherichia coli involved in aminosugar catabolism, has been crystallized by the vapour-diffusion technique using phosphate as precipitant. X-ray diffraction experiments show the crystals to belong to the orthorhombic crystal system, with space group P21212. The unit-cell parameters are a = 82.09,(2), b = 114.50,(1), c = 80.17,(1),Å. The crystals diffract to a maximum resolution of 1.8,Å and an initial data set was collected to 2.0,Å. [source]

Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010
Francieli Colussi
The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source. [source]