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Native Data Set (native + data_set)
Kinds of Native Data Set Selected AbstractsCrystallization and preliminary X-ray crystallographic studies on the bacteriophage ,6 RNA-dependent RNA polymeraseACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2000Sarah J. Butcher The RNA-dependent RNA polymerase (P2) from bacteriophage ,6 has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. A fully substituted selenomethionyl version of the protein has also been produced. Crystals of both proteins have been grown; most belong to the monoclinic space group P21, with unit-cell parameters a = 105.9, b = 94.0, c = 140.9,Å, , = 101.4°, but some are trigonal (space group P31 or P32), with unit-cell parameters a = b = 110.1, c = 159.4,Å, , = 120°. Both crystal forms occur in the same crystallization drop and are morphologically indistinguishable. Native data sets have been collected from both types of crystals to better than 3,Å resolution. [source] Structural studies of MIP synthaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2000Adam J. Stein The conversion of glucose 6-phosphate to 1- l - myo -inositol 1-phosphate (MIP) by 1- l - myo -inositol 1-phosphate synthase (MIP synthase) is the first committed and rate-limiting step in the de novo biosynthesis of inositol in all eukaryotes. The importance of inositol-containing molecules both as membrane components and as critical second messenger signal-transduction species make the function and regulation of this enzyme important for a host of biologically important cellular functions including proliferation, neurostimulation, secretion and contraction. MIP synthase has been overexpressed in Esherichia coli and purified to homogeneity by chromatographic methods. Two crystal forms of MIP synthase were obtained by the hanging-drop vapor-diffusion method. Native data sets for both crystal forms were collected in-house on a Rigaku R-AXIS IIC imaging-plate detector. Crystal form I belongs to space group C2, with unit-cell parameters a = 153.0, b = 96.6, c = 122.6,Å, , = 126.4°, and diffracts to 2.5,Å resolution. Crystal form II belongs to space group P21, with unit-cell parameters a = 94.5, b = 186.2, c = 86.5,Å, , = 110.5°, and diffracts to 2.9,Å resolution. [source] Crystallization and preliminary X-ray diffraction studies of vitamin D3 hydroxylase, a novel cytochrome P450 isolated from Pseudonocardia autotrophicaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Yoshiaki Yasutake Vitamin D3 hydroxylase (Vdh) is a novel cytochrome P450 monooxygenase isolated from the actinomycete Pseudonocardia autotrophica and consisting of 403 amino-acid residues. Vdh catalyzes the activation of vitamin D3via sequential hydroxylation reactions: these reactions involve the conversion of vitamin D3 (cholecalciferol or VD3) to 25-hydroxyvitamin D3 [25(OH)VD3] and the subsequent conversion of 25(OH)VD3 to 1,,25-dihydroxyvitamin D3 [calciferol or 1,,25(OH)2VD3]. Overexpression of recombinant Vdh was carried out using a Rhodococcus erythropolis expression system and the protein was subsequently purified and crystallized. Two different crystal forms were obtained by the hanging-drop vapour-diffusion method at 293,K using polyethylene glycol as a precipitant. The form I crystal belonged to the trigonal space group P31, with unit-cell parameters a = b = 61.7, c = 98.8,Å. There is one Vdh molecule in the asymmetric unit, with a solvent content of 47.6%. The form II crystal was grown in the presence of 25(OH)VD3 and belonged to the orthorhombic system P212121, with unit-cell parameters a = 63.4, b = 65.6 c = 102.2,Å. There is one Vdh molecule in the asymmetric unit, with a solvent content of 46.7%. Native data sets were collected to resolutions of 1.75 and 3.05,Å for form I and form II crystals, respectively, using synchrotron radiation. The structure solution was obtained by the molecular-replacement method and model refinement is in progress for the form I crystal. [source] Crystallization and preliminary X-ray analysis of a C-terminal TonB fragment from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2004Jiri Koedding The TonB protein located in the cell wall of Gram-negative bacteria mediates the proton motive force from the cytoplasmic membrane to specific outer membrane transporters. A C-terminal fragment of TonB from Escherichia coli consisting of amino-acid residues 147,239 (TonB-92) has been purified and crystallized. Crystals grew in space group P21 to dimensions of about 1.0 × 0.12 × 0.12,mm. A native data set has been obtained to 1.09,Å resolution. [source] Crystallization and preliminary crystallographic study of the functional form of the Bacillus thuringiensis mosquito-larvicidal Cry4Aa mutant toxinACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2004Panadda Boonserm The 65,kDa functional form of the mosquito-larvicidal Cry4Aa-R235Q mutant toxin has been crystallized. The crystals belong to space group C2221, with unit-cell parameters a = 91.2, b = 202.1, c = 98.7,Å, and contain one molecule per asymmetric unit. The crystals diffract to ,2.9,Å using synchrotron radiation and a complete native data set has been collected. The structure has been solved using a molecular-replacement method with the Cry4Ba toxin protein as a search model. [source] Crystallization and preliminary crystallographic analysis of a novel haemolytic lectin from the mushroom Laetiporus sulphureusACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2004José 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] Crystallization and preliminary X-ray analysis of a novel Trichoderma reesei xylanase IV belonging to glycoside hydrolase family 5ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Tarja Parkkinen Xylanase IV (XYN IV) is a new recently characterized xylanase from Trichoderma reesei. It is able to degrade several different xylans, mainly producing xylose. XYN IV has been crystallized by the hanging-drop vapour-diffusion method, using PEG 6000 as a precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 86.3, b = 137.5, c = 196.1,Å, , = , = , = 90°. Assuming a molecular weight of 50.3,kDa, the VM values indicate there to be four XYN IV monomers in an asymmetric unit and the solvent content of the crystals to be 57%. Based on dynamic light-scattering measurements, XYN IV is a dimer in solution. A native data set to 2.8,Å resolution has been collected at a home laboratory and a data set to 2.2,Å resolution has been collected using synchrotron radiation. [source] Purification, crystallization and preliminary X-ray analysis of a water-soluble chlorophyll protein from Brassica oleracea L. var. acephala (kale)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Daisuke Horigome A water-soluble chlorophyll protein (WSCP) with a chlorophyll a:b ratio of 6:1 from Brassica oleracea L. var. acephala (kale) was purified and crystallized by the hanging-drop vapour-diffusion method using PEG 8000 and zinc acetate as precipitants. The crystal belongs to the hexagonal space group P6422, with unit-cell parameters a = b = 162.2, c = 38.7,Å. A native data set was collected to 2.80,Å resolution at 293,K using Cu,K, radiation from a rotating-anode generator. Preliminary analysis via molecular replacement identified one kale WSCP monomer in the asymmetric unit. The crystal packing showed a tetrameric structure for kale WSCP, as suggested by previous biochemical studies of WSCPs from Brassicaceae plants. [source] Crystallization and preliminary X-ray analysis of Yersinia pseudotuberculosis -derived mitogenACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2003Roberta Donadini Yersinia pseudotuberculosis -derived mitogen (YPM), a superantigen with no amino-acid sequence similarity to other known superantigens, has been crystallized by the sitting-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 138.67, b = 78.66, c = 32.91,Å, , = 91.97°. A native data set has been collected to a resolution of 1.8,Å using synchrotron radiation. Self-rotation function calculations suggest the presence of three molecules in the asymmetric unit, corresponding to a solvent content of 45%. [source] Crystallization and preliminary X-ray studies of the glutaredoxin from poplar in complex with glutathioneACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2003Katia D'Ambrosio A monocysteinic mutant of poplar glutaredoxin (C30S) has been overproduced and purified. The protein has been crystallized in complex with glutathione using the hanging-drop vapour-diffusion technique in the presence of PEG 4000 as a precipitating agent. A native data set was collected at 1.55,Å resolution. The crystals belong to space group P212121, with unit-cell parameters a = 45.7, b = 49.1, c = 104.8,Å. Isomorphous crystals of a selenomethionine derivative were grown under the same conditions. Three data sets were collected at 1.73,Å using the FIP synchrotron beamline at the ESRF. The positions of the Se atoms were determined and model rebuilding and refinement are in progress. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of HP1352, a putative DNA methyltransferase in Helicobacter pyloriACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2003Katja Schirwitz The putative methyltransferase gene HP1352 from Helicobacter pylori strain 26695 was heterologously expressed in Escherichia coli. The 359-amino-acid gene product was purified and crystallized. The crystals belong to space group I212121 and show diffraction to at least 2.5,Å resolution. The unit-cell parameters are a = 69.6, b = 86.6, c = 140.0,Å. A greater than 90% complete native data set has been collected and structure determination using the molecular-replacement method is ongoing. [source] Crystallization and preliminary X-ray diffraction analysis of recombinant hydrolase domain of 10-formyltetrahydrofolate dehydrogenaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 10-2 2002Alexander A. Chumanevich 10-Formyltetrahydrofolate dehydrogenase (FDH) is an abundant enzyme in liver cytosol. It is important for the regulation of 10-formyltetrahydrofolate/tetrahydrofolate pools, for de novo purine biosynthesis and for the removal of formate in the form of CO2. The enzyme is a natural fusion of two unrelated genes and consists of two functional catalytic domains. Here, the crystallization of the N-terminal domain of FDH is reported. This domain binds folate and functions as a 10-formyltetrahydrofolate hydrolase. The crystals grow as either spear-shaped needles or large plates, with the largest crystals reaching dimensions of 1.2 × 0.2 × 0.05,mm. Diffraction analysis revealed the space group to be P21212, with unit-cell parameters a = 100.00, b = 64.63, c = 64.59,Å. Based on the estimated solvent content, there is one 34,kDa molecule in the asymmetric unit. A native data set extending to 2.3,Å resolution has been collected with good merging statistics. [source] Crystallization and preliminary X-ray diffraction studies of catalase,peroxidase from Synechococcus PCC 7942ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2002Kei Wada The recombinant catalase,peroxidase of Synechococcus PCC 7942 overexpressed in Escherichia coli was purified and crystallized by the hanging-drop vapour-diffusion method using sodium formate as a precipitant. The crystals belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 109.3, c = 202.0,Å. The calculated VM value based on a dimer in the asymmetric unit was 1.9,Å3,Da,1. A native data set was collected to 2.3,Å resolution from a frozen crystal using synchrotron radiation at SPring-8. [source] Crystallization of quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni: crystals with unique optical propertiesACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001Arthur Oubrie Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni is a functional electron-transfer protein containing both a haem c and a pyrroloquinoline quinone cofactor. The enzyme has been crystallized at 277,K using polyethylene glycol 6000 as precipitant. The crystals belong to space group C2, with unit-cell parameters a = 98.1, b = 74.3, c = 92.2,Å, , = 105.9°. A native data set with a resolution of 2.44,Å resolution has been collected. The approximate orientation of the haem group with respect to the unit-cell axes has been determined from the optical properties of the crystals. [source] Structure of lobster apocrustacyanin A1 using softer X-raysACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001M. Cianci The molecular basis of the camouflage colouration of marine crustacea is often provided by carotenoproteins. The blue colour of the lobster carapace, for example, is intricately associated with a multimacromolecular 16-mer complex of protein subunits each with a bound astaxanthin molecule. The protein subunits of crustacyanin fall into two distinct subfamilies, CRTC and CRTA. Here, the crystal structure solution of the A1 protein of the CRTC subfamily is reported. The problematic nature of the structure solution of the CRTC proteins (both C1 and A1) warranted consideration and the development of new approaches. Three putative disulfides per protein subunit were likely to exist based on molecular-homology modelling against known lipocalin protein structures. With two such subunits per crystallographic asymmetric unit, this direct approach was still difficult as it involved detecting a weak signal from these sulfurs and suggested the use of softer X-rays, combined with high data multiplicity, as reported previously [Chayen et al. (2000), Acta Cryst. D56, 1064,1066]. This paper now describes the structure solution of CRTC in the form of the A1 dimer based on use of softer X-rays (2,Å wavelength). The structure solution involved a xenon derivative with an optimized xenon LI edge signal and a native data set. The hand of the xenon SIROAS phases was determined by using the sulfur anomalous signal from a high-multiplicity native data set also recorded at 2,Å wavelength. For refinement, a high-resolution data set was measured at short wavelength. All four data sets were collected at 100,K. The refined structure to 1.4,Å resolution based on 60,276 reflections has an R factor of 17.7% and an Rfree of 22.9% (3137 reflections). The structure is that of a typical lipocalin, being closely related to insecticyanin, to bilin-binding protein and to retinol-binding protein. This A1 monomer or dimer can now be used as a search motif in the structural studies of the oligomeric forms ,- and ,-crustacyanins, which contain bound astaxanthin molecules. [source] Crystallization and preliminary X-ray analysis of a thermoalkalophilic lipase from Bacillus stearothermophilus L1ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001Seong-Tae Jeong A thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) was crystallized in two different crystal forms using a low concentration of the enzyme and a calcium-exchange process. The first, needle-like, crystal form, which diffracts to about 3.5,Å, belongs to the orthorhombic space group P212121, with unit-cell parameters a = 67.84, b = 72.96, c = 104.41,Å. The second, monoclinic, crystal form, which behaves better than the first form for crystallographic analyses, belongs to the monoclinic space group C2 and has unit-cell parameters a = 119.62, b = 85.05, c = 98.36,Å, , = 99.73°. From the monoclinic crystals, a native data set and a samarium-derivative data set were collected to 2.0 and 2.3,Å resolution, respectively. The difference Patterson map between the two data sets shows strong heavy-atom peaks, indicating that the crystals are suitable for a high-resolution structure determination. [source] Crystallization and preliminary X-ray diffraction analysis of Thermus thermophilus prolyl-tRNA synthetaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2000Anna Yaremchuk Prolyl-tRNA synthetase from Thermus thermophilus (ProRSTT) was purified to homogeneity using a five-step purification procedure and was crystallized using ethylene glycol as a precipitant. Crystals of ProRSTT belong to the space group P21212, with unit-cell parameters a = 132, b = 191, c = 125,Å, have two homodimers per asymmetric unit and diffract to 2.4,Å resolution. A complete native data set to 2.43,Å resolution has been collected and a data set from ProRSTT in complex with proline has been collected to 2.9,Å resolution. [source] Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of human coronavirus OC43 nucleocapsid proteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010I-Jung Chen The N-terminal domain of nucleocapsid protein from human coronavirus OC43 (HCoV-OC43 N-NTD) mostly contains positively charged residues and has been identified as being responsible for RNA binding during ribonucleocapsid formation in the coronavirus. In this study, the crystallization and preliminary crystallographic analysis of HCoV-OC43 N-NTD (amino acids 58,195) with a molecular weight of 20,kDa are reported. HCoV-OC43 N-NTD was crystallized at 293,K using PEG 1500 as a precipitant and a 99.9% complete native data set was collected to 1.7,Å resolution at 100,K with an overall Rmerge of 5.0%. The crystals belonged to the hexagonal space group P65, with unit-cell parameters a = 81.57, c = 42.87,Å. Solvent-content calculations suggest that there is likely to be one subunit of N-NTD in the asymmetric unit. [source] Purification, crystallization and preliminary X-ray diffraction analysis of the lytic transglycosylase MltF from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Pramod K. Madoori The lytic transglycosylase MltF from Escherichia coli is an outer-membrane-bound periplasmic protein with two domains: a C-terminal catalytic domain with a lysozyme-like fold and an N-terminal domain of unknown function that is homologous to the periplasmic substrate-binding proteins of ABC transporters. In order to investigate its structure and function, a soluble form of full-length MltF (sMltF) containing both domains and a soluble fragment containing only the N-terminal domain (sMltF-NTD) were purified and crystallized. Crystals of sMltF belonged to space group P43212 or P41212, with unit-cell parameters a = b = 110.8, c = 163.5,Å and one or two molecules per asymmetric unit. A complete data set was collected to 3.5,Å resolution. Crystals of sMltF-NTD belonged to space group P3121, with unit-cell parameters a = b = 82.4, c = 75.2,Å and one molecule per asymmetric unit. For sMltF-NTD, a complete native data set was collected to 2.20,Å resolution. In addition, for phasing purposes, a three-wavelength MAD data set was collected to 2.5,Å resolution using a bromide-soaked sMltF-NTD crystal. Using phases derived from the Br-MAD data, it was possible to build a partial model of sMltF-NTD. [source] Crystallization and preliminary X-ray crystallographic analysis of a GroEL1 fragment from Mycobacterium tuberculosis H37RvACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Bernhard Sielaff Full-length GroEL1 from Mycobacterium tuberculosis H37Rv was cloned, overexpressed and purified. Crystals were obtained by the hanging-drop vapor-diffusion method and contained a 23,kDa GroEL1 fragment. A complete native data set was collected from a single frozen crystal that belonged to the orthorhombic space group P21212, with unit-cell parameters a = 75.47, b = 78.67, c = 34.89,Å, , = , = , = 90°, and diffracted to 2.2,Å resolution on a home X-ray source. [source] Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Ulrika W. Hultdin The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein,protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His6 -tagged fusion protein was captured by Ni2+ -affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293,K and that removal of the highly flexible His6 tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295,K. A native data set to 2.0,Å resolution was collected at 100,K using synchrotron radiation. [source] Crystallization and preliminary X-ray analysis of Psu, an inhibitor of the bacterial transcription terminator RhoACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Susmita Khamrui Psu, a coat protein from bacteriophage P4, inhibits Rho-dependent transcription termination both in vivo and in vitro. The Psu protein is ,-helical in nature and appeared to be a dimer in solution. It interacts with Rho and affects the ATP binding and RNA-dependent ATPase activity of Rho, which in turn reduces the rate of RNA release from the elongation complex. Crystals of Psu were grown in space group I422 in the presence of PEG, with unit-cell parameters a = b = 148.76, c = 63.38,Å and a calculated Matthews coefficient of 2.1,Å3,Da,1 (41.5% solvent content), assuming the presence of two molecules in the asymmetric unit. A native data set was collected to 2.3,Å resolution. [source] Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 domain bound to a palindromic SBE DNA elementACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Nithya Baburajendran The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7,Å resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2,M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78,Å, , = , = , = 90°. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit. [source] Crystallization and preliminary X-ray diffraction analysis of motif N from Saccharomyces cerevisiae Dbf4ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Lindsay A. Matthews The Cdc7,Dbf4 complex plays an instrumental role in the initiation of DNA replication and is a target of replication-checkpoint responses in Saccharomyces cerevisiae. Cdc7 is a conserved serine/threonine kinase whose activity depends on association with its regulatory subunit, Dbf4. A conserved sequence near the N-terminus of Dbf4 (motif N) is necessary for the interaction of Cdc7,Dbf4 with the checkpoint kinase Rad53. To understand the role of the Cdc7,Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. A complete native data set was collected at 100,K from crystals that diffracted X-rays to 2.75,Å resolution and structure determination is currently under way. [source] Crystallization and preliminary X-ray diffraction studies of the tetramerization domain derived from the human potassium channel Kv1.3ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Andreas Winklmeier The tetramerization domain (T1 domain) derived from the voltage-dependent potassium channel Kv1.3 of Homo sapiens was recombinantly expressed in Escherichia coli and purified. The crystals were first grown in an NMR tube in 150,mM potassium phosphate pH 6.5 in the absence of additional precipitants. The crystals showed I4 symmetry characteristic of the naturally occurring tetrameric assembly of the single subunits. A complete native data set was collected to 1.2,Å resolution at 100,K using synchrotron radiation. [source] Purification, crystallization and preliminary X-ray diffraction analysis of a plant subtilaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Rolf Rose The subtilase SBT3 from Solanum lycopersicum (tomato) was purified from a tomato cell culture and crystallized using the sitting-drop vapour-diffusion method. A native data set was collected to 2.5,Å resolution at 100,K using synchrotron radiation. For experimental phasing, CsCl-derivative and tetrakis(acetoxymercuri)methane (TAMM) derivative crystals were employed for MIRAS phasing. Three caesium sites and one TAMM site were identified, which allowed solution of the structure. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of YvoA from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Marcus Resch The putative transcriptional regulator protein YvoA (BSU35030) from Bacillus subtilis was cloned and heterologously expressed in Escherichia coli. The protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography and subsequently crystallized. A complete native data set was collected to 2.50,Å resolution. The crystals belonged to the monoclinic space group C2 and preliminary analysis of the diffraction data indicated the presence of approximately 12 molecules per asymmetric unit. [source] Crystallization and preliminary crystallographic analysis of thermophilic cellulase from Fervidobacterium nodosum Rt17-B1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Baisong Zheng FnCel5A, a thermostable endoglucanase, is a member of glycohydrolase family 5 which catalyzes the hydrolysis of cellulose to glucose in the thermophilic bacterium Fervidobacterium nodosum Rt17-B1. FnCel5A is particularly interesting because of its high thermostability (Topt = 353,K, half-life 48,h) and its high specific activity towards carboxymethylcellulose. These properties make FnCel5A an attractive target for protein engineering to improve cellulase activity. In order to resolve the crystal structure of FnCel5A and to gain a better understanding of its biological function, recombinant FnCel5A was expressed, purified and crystallized at 291,K using NaH2PO4/KH2PO4 as a precipitant. A 2.4,Å resolution native data set was collected from a single flash-cooled crystal (100,K) using 20%(v/v) glycerol as a cryoprotectant. These crystals belonged to space group P21212, with unit-cell parameters a = 53.5, b = 81.7, c = 85.2,Å, , = , = , = 90°. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.5,Å3,Da,1. A data set was also collected to 1.7,Å resolution from a selenomethionyl derivative; it belonged to space group P212121 with the same unit-cell parameters as the native crystals. [source] Purification, crystallization and preliminary X-ray diffraction studies of a putative UDP- N -acetyl- d -mannosamine dehydrogenase from Pyrococcus horikoshii OT3ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2007Neratur K. Lokanath A putative UDP- N -acetyl- d -mannosamine dehydrogenase from Pyrococcus horikoshii OT3, an essential enzyme for polysaccharide biosynthesis, has been overexpressed in Escherichia coli and purified. Crystals were obtained using the oil-microbatch method at 291,K. A native data set extending to 1.8,Å resolution has been collected and processed in space group P21. Assuming the presence of a dimer in the asymmetric unit, the VM value is calculated to be 2.3,Å3,Da,1, which is consistent with the result of a dynamic light-scattering experiment that shows a dimeric state of the protein in solution. [source] Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2007Katsumi Shimizu Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide-synthesis salvage pathway. In order to study the structure,thermostability relationship of this enzyme, PYNP from the extreme thermophile Thermus thermophilus HB8 (TTHA1771) has been cloned, overexpressed and purified. The TTHA1771 protein was crystallized at 291,K using the oil-microbatch method with PEG 4000 as a precipitant. A native data set was collected to 1.8,Å resolution using synchrotron radiation. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 58.83, b = 76.23, c = 103.86,Å, , = 91.3°. [source] | ||||||||||