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Diffraction Data Set (diffraction + data_set)
Kinds of Diffraction Data Set Selected AbstractsCrystallization and preliminary X-ray analysis of human endonuclease 1 (APE1) in complex with an oligonucleotide containing a 5,6-dihydrouracil (DHU) or an ,-anomeric 2,-deoxyadenosine (,dA) modified baseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Pascal Retailleau The multifunctional human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a key enzyme involved in both the base-excision repair (BER) and nucleotide-incision repair (NIR) pathways. In the NIR pathway, APE1 incises DNA 5, to a number of oxidatively damaged bases. APE1 was crystallized in the presence of a 15-mer DNA containing an oxidatively damaged base in a single central 5,6-dihydrouracil (DHU)·T or ,-anomeric 2,-deoxyadenosine (,dA)·T base pair. Diffraction data sets were collected to 2.2 and 2.7,Å resolution from DNA-DHU,APE1 and DNA-,dA,APE1 crystals, respectively. The crystals were isomorphous and contained one enzyme molecule in the asymmetric unit. Molecular replacement was performed and the initial electron-density maps revealed that in both complexes APE1 had crystallized with a degradation DNA product reduced to a 6-mer, suggesting that NIR and exonuclease reactions occurred prior to crystallization. [source] Crystallization and preliminary crystallographic analysis of the catalytic module of endolysin from Cp-7, a phage infecting Streptococcus pneumoniaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Noella Silva-Martin As part of the life cycle of the pneumococcal phage Cp-7, the endolysin Cpl-7 cleaves the glycosidic ,1,4 bonds between N -acetylmuramic acid and N -acetylglucosamine in the pneumococcal cell wall, resulting in bacterial lysis. Recombinant Cpl-7 was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method at 291,K. Diffraction-quality tetragonal crystals of the catalytic module of Cpl-7 were obtained from a mixture of PEG 3350 and sodium formate. The crystals belonged to space group I422, with unit-cell parameters a = 127.93, b = 127.93, c = 82.07,Å. Diffraction data sets were collected to 2.4,Å resolution using a rotating-anode generator. [source] Expression, purification, crystallization and preliminary X-ray analysis of rice (Oryza sativa L.) Os4BGlu12 ,-glucosidaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Sompong Sansenya Rice (Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 ,-glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N-terminal thioredoxin/His6 tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N-terminal tag. Native Os4BGlu12 and its complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-,- d -glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1,M Tris,HCl pH 8.5, 0.16,M NaCl at 288,K. Diffraction data sets for the apo and inhibitor-bound forms were collected to 2.50 and 2.45,Å resolution, respectively. The space group and the unit-cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P43212 by molecular replacement using the white clover cyanogenic ,-glucosidase structure (PDB code 1cbg) as a search model. [source] Production, crystallization and preliminary crystallographic analysis of an exosite-containing fragment of human von Willebrand factor-cleaving proteinase ADAMTS13ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Masashi Akiyama ADAMTS13 is a reprolysin-type metalloproteinase belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motif) family. It specifically cleaves plasma von Willebrand factor (VWF) and regulates platelet adhesion and aggregation. ADAMTS13 is a multi-domain enzyme. In addition to the N-terminal metalloproteinase domain, the ancillary domains, including a disintegrin-like domain, a thrombospondin-1 type 1 repeat, a Cys-rich domain and a spacer domain, are required for VWF recognition and cleavage. In the present study, a fragment of the ADAMTS13 ancillary domains (ADAMTS13-DTCS; residues 287,685) was expressed using CHO Lec cells, purified and crystallized. Diffraction data sets were collected using the SPring-8 beamline. Two ADAMTS13-DTCS crystals with distinct unit-cell parameters generated data sets to 2.6 and 2.8,Å resolution, respectively. [source] Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2006D. Aragão The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65,Å. The phase problem was solved for a P21 crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P21 has unit-cell parameters a = 105.5, b = 85.7, c = 151.8,Å, , = 105.2°. Model building and refinement are currently under way. [source] The minimum crystal size needed for a complete diffraction data setACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010James M. Holton In this work, classic intensity formulae were united with an empirical spot-fading model in order to calculate the diameter of a spherical crystal that will scatter the required number of photons per spot at a desired resolution over the radiation-damage-limited lifetime. The influences of molecular weight, solvent content, Wilson B factor, X-ray wavelength and attenuation on scattering power and dose were all included. Taking the net photon count in a spot as the only source of noise, a complete data set with a signal-to-noise ratio of 2 at 2,Å resolution was predicted to be attainable from a perfect lysozyme crystal sphere 1.2,µm in diameter and two different models of photoelectron escape reduced this to 0.5 or 0.34,µm. These represent 15-fold to 700-fold less scattering power than the smallest experimentally determined crystal size to date, but the gap was shown to be consistent with the background scattering level of the relevant experiment. These results suggest that reduction of background photons and diffraction spot size on the detector are the principal paths to improving crystallographic data quality beyond current limits. [source] Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2009Soheila Emamzadah Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2,mm thin transparent cards which contain 500 chambers, each with a volume of 320,nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus' molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5,Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts. [source] Purification, crystallization and preliminary X-ray analysis of Triatoma virus (TrV) from Triatoma infestansACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004Gabriela 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 crystallographic analysis of endonuclease VIII in its uncomplexed formACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004Gali 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] A new crystal form of XT6 enables a significant improvement of its diffraction quality and resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Maya Bar Xylanases (1,4-,- d -xylan xylanhydrolases; EC 3.2.1.8) hydrolyze the 1,4-,- d -xylopyranosyl linkage of xylans. The detailed structural characterization of these enzymes is of interest for the elucidation of their catalytic mechanism and for their rational modification toward improved stability and specificity. An extracellular xylanase from Geobacillus stearothermophilus T-6 (XT6) has recently been cloned, overexpressed, purified and biochemically characterized. Previous crystallographic efforts resulted in a hexagonal crystal form, which subsequently proved to be of limited use for structural analysis, mainly because of its relatively poor diffraction quality and resolution. A systematic search for more suitable crystals of XT6 recently resulted in a new crystal form of this enzyme with significantly improved diffraction characteristics. The new crystals belong to a C -centred monoclinic crystal system (space group C2), with unit-cell parameters a = 121.5, b = 61.7, c = 89.1,Å, , = 119.7°. These crystals diffract X-rays to better than 1.5,Å resolution, showing a very clear diffraction pattern of relatively high quality. The crystals are mechanically strong and exhibit excellent radiation-stability when frozen under cold nitrogen gas. A full diffraction data set to 1.45,Å resolution (94.1% completeness, Rmerge = 7.0%) has been collected from flash-frozen crystals of the native enzyme at 95,K using synchrotron radiation. Crystals of the E159A/E265A catalytic double mutant of XT6 were found to be isomorphous to those of native XT6. They were used for a full measurement of 1.8,Å resolution diffraction data at 100,K (90.9% completeness; Rmerge = 5.0%). These data are currently being used for the high-resolution structure determination of XT6 and its mutant for mechanistic interpretations and rational introduction of thermostability. [source] Crystallization of the oligopeptide-binding protein AppA from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004AppA is the membrane-anchored extracellular receptor component of an ABC transporter responsible for the uptake of oligopeptides into Bacillus subtilis. AppA has been overexpressed as a cleavable maltose-binding protein fusion in Escherichia coli. Following removal of the fusion portion, AppA has been crystallized from morpholinoethanesulfonic acid-buffered solutions at pH 6.5 containing polyethylene glycol and zinc acetate. A complete X-ray diffraction data set extending to 2.3,Å spacing has been collected. [source] Purification, crystallization and X-ray diffraction analysis of the extracellular part of the human Fc receptor for IgA, Fc,RI (CD89)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Katja Wenig Fc,RI is the predominant receptor for IgA in the serum. Nevertheless, the interaction between the molecules that finally leads to an immune response is poorly understood. To investigate the structural requirements for IgA binding, the extracellular region of Fc,RI was cloned and overexpressed in Escherichia coli. The resulting inclusion-body protein was refolded and purified. Despite its deglycosylated state, this recombinant Fc,RI retained its ability to bind human IgA. The protein crystallized spontaneously as microcrystalline needles. Recrystallization yielded crystals belonging to a primitive monoclinic space group. A complete 2.8,Å resolution X-ray diffraction data set was collected using synchrotron radiation. [source] Expression, purification, crystallization and preliminary X-ray crystallographic studies of a psychrophilic cellulase from Pseudoalteromonas haloplanktisACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2003Sébastien Violot The Antarctic psychrophile Pseudoalteromonas haloplanktis produces a cold-active cellulase. To date, a three-dimensional structure of a psychrophilic cellulase has been lacking. Crystallographic studies of this cold-adapted enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of the cold adaptation and the high catalytic efficiency of the enzyme at low and moderate temperatures. The catalytic core domain of the psychrophilic cellulase CelG from P. haloplanktis has been expressed, purified and crystallized and a complete diffraction data set to 1.8,Å has been collected. The space group was found to be P212121, with unit-cell parameters a = 135.1, b = 78.4, c = 44.1,Å. A molecular-replacement solution, using the structure of the mesophilic counterpart Cel5A from Erwinia chrysanthemi as a search model, has been found. [source] Crystallization of the 43,kDa ATPase domain of Thermus thermophilus gyrase B in complex with novobiocinACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2002V. Lamour The 43,kDa ATPase domain of Thermus thermophilus gyrase B was overproduced in Escherichia coli and a three-step purification protocol yielded large quantities of highly purified enzyme which remained stable for weeks. Crystals of the 43,kDa domain in complex with novobiocin, one of the most potent inhibitors of bacterial topoisomerases, were obtained. Crystals obtained in the presence of PEG 8000 do not diffract, but a different crystal form was obtained using sodium formate as a precipitating agent. The plate-shaped crystals, which were less than 10,µm in thickness, could be cryocooled directly from the mother liquor and a full diffraction data set was collected to 2.3,Å allowing the determination of the first structure of a gyrase B 43K domain in complex with a coumarin. [source] Crystallization and preliminary X-ray analysis of the complex of porcine pancreatic elastase and a hybrid squash inhibitorACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002Kai Hilpert A hybrid inhibitor consisting of the scaffold of a squash-type inhibitor and a specific inhibitory peptide optimized from the third domain of ovomucoid inhibitor from turkey against porcine pancreatic elastase was synthesized by peptide synthesis. The complex formed by this hybrid inhibitor and the porcine pancreatic elastase was crystallized using the hanging-drop method with citrate in the crystallization solution. The space group was determined to be P212121, with unit-cell parameters a = 56.33, b = 56.44, c = 72.76,Å. A complete X-ray diffraction data set was collected under cryogenic conditions to 1.8,Å. [source] Multiple isomorphous replacement on merohedral twins: structure determination of deacetoxycephalosporin C synthaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2001Anke C. Terwisscha van Scheltinga Merohedral twinning is a packing anomaly that seriously impairs the determination of macromolecular crystal structures. Crystals of deacetoxycephalosporin C synthase (DAOCS), an enzyme involved in the expansion of the penicillin nucleus to form the core structure of the cephalosporin antibiotics, were found to be merohedrally twinned by many diagnostic criteria. Here, the structure determination of DAOCS from twinned crystals based on a combination of isomorphous replacement and the use of a multiple-wavelength diffraction data set is described. [source] Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeriACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2001Xavier Carpena Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P21, with unit-cell parameters a = 60.6, b = 153.9, c = 109.2,Å, , = 102.8°. From these crystals a diffraction data set to 1.8,Å resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I212121), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5,Å. These crystals diffracted beyond 2.2,Å resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis. [source] Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Francieli 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] Cloning, expression, purification, crystallization and preliminary crystallographic analysis of 5-aminolaevulinic acid dehydratase from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Qianda Lu 5-Aminolaevulinic acid dehydratase (ALAD), a crucial enzyme in the biosynthesis of tetrapyrrole, catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PBG). The gene encoding ALAD was amplified from genomic DNA of Bacillus subtilis and the protein was overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified and crystallized with an additional MGSSHHHHHHSSGLVPRGSH, tag at the N-terminus of the target protein. Diffraction-quality single crystals were obtained by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at a resolution of 2.7,Å. [source] Crystallization and preliminary X-ray analysis of a glucansucrase from the dental caries pathogen Streptococcus mutansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Keisuke Ito Glucansucrases encoded by Streptococcus mutans play essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI from S. mutans was overexpressed in Escherichia coli strain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space group P21212 were obtained. A diffraction data set was collected to 2.1,Å resolution. [source] Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Jiang Gu Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a ,-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7,Å resolution. The crystals belonged to space group I4132, with unit-cell parameter a = 158.99,Å. The Matthews coefficient and solvent content were calculated to be 2.55,Å3,Da,1 and 51.77%, respectively, for two molecules in the asymmetric unit. [source] Expression, purification and crystallization of a plant type III polyketide synthase that produces diarylheptanoidsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Hiroyuki Morita Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase that catalyzes the one-pot formation of bisdemethoxycurcumin by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. Recombinant CUS was expressed in Escherichia coli and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 72.7, b = 97.2, c = 126.2,Å, , = , = 90.0, , = 103.7°. A diffraction data set was collected in-house to 2.5,Å resolution. [source] Crystallization and preliminary X-ray diffraction analysis of the putative aldose 1-epimerase YeaD from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Weijie You Escherichia coli YeaD (ecYeaD) is suggested to be a member of the galactose mutarotase-like superfamily. Galactose mutarotase is an enzyme that converts ,-galactose to ,-galactose. The known structures of these galactose mutarotase-like proteins are similar to those of galactose mutarotases, with the catalytic residues being conserved, but there are some differences between them in the substrate-binding pocket. In order to reveal the specificity of ecYeaD, a three-dimensional structure is essential. Full-length ecYeaD with an additional 6×His tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283,K. An X-ray diffraction data set was collected to a resolution of 1.9,Å from a single flash-cooled crystal that belonged to space group P212121. [source] Crystallization and preliminary diffraction analysis of an engineered cephalosporin acylaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Anandhi Anandan Crystallization conditions are reported for an engineered cephalosporin acylase based on the sequence of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas strain N176. Initial crystals were grown using polyethylene glycol as a crystallizing agent; however, these crystals diffracted poorly and exhibited high mosaicity. A dehydration procedure in which crystals were transferred to a solution containing a higher concentration of polyethylene glycol as well as glycerol improved the diffraction quality such that a 1.57,Å diffraction data set could be obtained. [source] Fab crystallization and preliminary X-ray analysis of NC-1, an anti-HIV-1 antibody that recognizes the six-helix bundle core of gp41ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Lei Jin NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3221, with unit-cell parameters a = b = 118.7, c = 106.0,Å. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2,Å resolution and a clear molecular-replacement solution was obtained for solution of the structure. [source] Crystallization and preliminary crystallographic studies of a flavin-dependent thymidylate synthase from Helicobacter pyloriACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Xiaoli Zhang The ThyX enzymes that have recently been identified in various bacteria, including some important human pathogens such as Helicobacter pylori and Mycobacterium tuberculosis, are flavin-dependent thymidylate synthases that function in the place of classic thymidylate synthase enzymes in the biosynthesis of dTMP, which is one of the building blocks of DNA. They are promising targets for the development of novel antibiotics because they utilize catalytic mechanisms that are distinct from those of the classic thymidylate synthases found in most organisms, including humans. In this study, H. pylori ThyX was purified and crystallized in complex with flavin adenine dinucleotide (FAD) and a diffraction data set was collected to 2.5,Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 221.92, b = 49.43, c = 143.02,Å, , = 98.84°. [source] Purification, crystallization and preliminary X-ray analysis of the aspartate aminotransferase of Plasmodium falciparumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Rishabh Jain Aspartate aminotransferases (EC 2.6.1.1) catalyse the conversion of aspartate and ,-ketoglutarate to oxaloacetate and glutamate in a reversible manner. Thus, the aspartate aminotransferase of Plasmodium falciparum (PfAspAT) plays a central role in the transamination of amino acids. Recent findings suggest that PfAspAT may also play a pivotal role in energy metabolism and the de novo biosynthesis of pyrimidines. While therapeutics based upon the inhibition of other proteins in these pathways are already used in the treatment of malaria, the advent of multidrug-resistant strains has limited their efficacy. The presence of PfAspAT in these pathways may offer additional opportunities for the development of novel therapeutics. In order to gain a deeper understanding of the function and role of PfAspAT, it has been expressed and purified to homogeneity. The successful crystallization of PfAspAT, the collection of a 2.8,Å diffraction data set and initial attempts to solve the structure using molecular replacement are reported. [source] Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Zhijie Li Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid ,-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12,mg,ml,1) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100,mM Tris,HCl pH 8.0, 150,mM sodium chloride, 200,mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8,Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3,Å, , = , = 90.0, , = 124.0°. A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient VM of 2.76,Å3,Da,1 and a solvent content of 55%. [source] Crystallization and preliminary X-ray crystallographic analysis of ,-galactosidase from Kluyveromyces lactisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Ángel Pereira-Rodríguez ,-Galactosidase from Kluyveromyces lactis catalyses the hydrolysis of the ,-galactosidic linkage in lactose. Owing to its many industrial applications, the biotechnological potential of this enzyme is substantial. This protein has been expressed in yeast and purified for crystallization trials. However, optimization of the best crystallization conditions yielded crystals with poor diffraction quality that precluded further structural studies. Finally, the crystal quality was improved using the streak-seeding technique and a complete diffraction data set was collected at 2.8,Å resolution. [source] Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Emmanuel Oluwadare Balogun In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His6 tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75,Å resolution indicated that the crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 63.84, b = 121.50, c = 154.59,Å. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (VM) of 2.5,Å3,Da,1, corresponding to 50% solvent content. [source] | ||||||||||