Home About us Contact
|
Home About us Contact | ||
|
|
||
Space Group P21212 (space + group_p21212)
Kinds of Space Group P21212 Selected AbstractsStructural features of a zinc binding site in the superantigen strepococcal pyrogenic exotoxin A (SpeA1): Implications for MHC class II recognitionPROTEIN SCIENCE, Issue 6 2001Matthew Baker Abstract Streptococcal pyrogenic exotoxin A (SpeA) is produced by Streptococcus pyogenes, and has been associated with severe infections such as scarlet fever and Streptococcal Toxic Shock Syndrome (STSS). In this study, the crystal structure of SpeA1 (the product of speA allele 1) in the presence of 2.5 mM zinc was determined at 2.8 Å resolution. The protein crystallizes in the orthorhombic space group P21212, with four molecules in the crystallographic asymmetric unit. The final structure has a crystallographic R -factor of 21.4% for 7,031 protein atoms, 143 water molecules, and 4 zinc atoms (one zinc atom per molecule). Four protein ligands,Glu 33, Asp 77, His 106, and His 110,form a zinc binding site that is similar to the one observed in a related superantigen, staphylococcoal enterotoxin C2. Mutant toxin forms substituting Ala for each of the zinc binding residues were generated. The affinity of these mutants for zinc ion confirms the composition of this metal binding site. The implications of zinc binding to SpeA1 for MHC class II recognition are explored using a molecular modeling approach. The results indicate that, despite their common overall architecture, superantigens appear to have multiple ways of complex formation with MHC class II molecules. [source] Determination of zinc incorporation in the Zn-substituted gallophosphate ZnULM-5 by multiple wavelength anomalous dispersion techniquesACTA CRYSTALLOGRAPHICA SECTION B, Issue 3 2010M. Helliwell The location of isomorphously substituted zinc over eight crystallographically different gallium sites has been determined in a single-crystal study of the gallophosphate ZnULM-5, GaZn(PO4)14(HPO4)2(OH)2F7, [H3N{CH2}6NH3]4, 6H2O, in an 11 wavelength experiment, using data from Station 9.8, SRS Daresbury. The measurement of datasets around the K edges of both Ga and Zn, as well as two reference datasets away from each absorption edge, was utilized to selectively exploit dispersive differences of each metal atom type in turn, which allowed the major sites of Zn incorporation to be identified as the metal 1 and 3 sites, M1 and M3. The preferential substitution of Zn at these sites probably arises because they are located in double four-ring (D4R) building units which can relax to accommodate the incorporation of hetero atoms. As the crystal is non-centrosymmetric, with space group P21212, it was also possible to use anomalous differences to corroborate the results obtained from the dispersive differences. These results were obtained firstly from difference Fourier maps, calculated using a phase set from the refined structure from data measured at the Zr K edge. Also, refined dispersive and anomalous occupancies, on an absolute scale, could be obtained using the program MLPHARE, allowing estimates for the Zn incorporation of approximately 22 and 18 at. % at the M1 and M3 sites to be obtained. In addition, f, and f,, values for Ga and Zn at each wavelength could be estimated both from MLPHARE results, and by refinement in JANA2006. The fully quantitative determinations of the dispersive and anomalous coefficients for Ga and Zn at each wavelength, as well as metal atom occupancies over the eight metal atom sites made use of the CCP4's MLPHARE program as well as SHELXL and JANA2006. The results by these methods agree closely, and JANA2006 allowed the ready determination of standard uncertainties on the occupancy parameters, which were for M1 and M3, 20.6,(3) and 17.2,(3),at %, respectively. [source] Diphenyldipyridinezinc(II): partial spontaneous resolution of an organometallic reagentACTA CRYSTALLOGRAPHICA SECTION C, Issue 5 2009Anders Lennartson The title compound, [Zn(C6H5)2(C5H5N)2], (I), forms conformationally chiral molecules residing on a twofold axis. The molecules are stacked along c, and these stacks are associated by edge-to-face ,,, interactions. Crystals of (I) belong to the Sohncke space group P21212 and the crystal lattice of (I) is chiral. The crystal batch that was examined consisted of a mixture of enantiomerically pure crystals and crystals twinned by inversion. [source] A case of structure determination using pseudosymmetryACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Sergei Radaev Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P21212. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P21, despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P21. Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P21212 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: am = bo,co, bm = ao and cm = ,2co,bo. The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement. [source] Ligand binding at the transthyretin dimer,dimer interface: structure of the transthyretin,T4Ac complex at 2.2,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2005Vivian Cody The crystal structure of the complex of human transthyretin (hTTR) with 3,3,,5,5,-tetraiodothyroacetic acid (T4Ac) has been determined to 2.2,Å resolution. The complex crystallizes in the orthorhombic space group P21212, with unit-cell parameters a = 43.46, b = 85.85, c = 65.44,Å. The structure was refined to R = 17.3% and Rfree = 21.9% for reflections without any ,-cutoff. T4Ac is bound in both the forward and the reverse mode in the two binding sites of hTTR. In the forward orientation, T4Ac binds in a position similar to that described for thyroxine (T4) in the orthorhombic hTTR,T4 complex. In this orientation, the iodine substituents of the phenolic ring are bound in the P3,/P2 halogen pockets. In the reverse orientation, which is the major binding mode of T4Ac, the ligand is bound deep in the TTR channel, with the carboxylic group bound in the P3, pocket and forming simultaneous polar interactions with the residues constituting the two hormone-binding sites. Such interactions of a thyroxine-analogue ligand bound in the reverse mode have never been observed in TTR complexes previously. [source] X-ray crystallographic studies of two transthyretin variants: further insights into amyloidogenesisACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2005Ricardo M. Neto-Silva Transthyretin (TTR) is a homotetrameric plasma protein that, as a result of a set of not yet fully characterized conformational changes, forms fibrillar aggregates that are the major protein component of amyloid deposits. More than 80 mutations associated with TTR amyloid deposition have been described in the literature. X-ray crystallography was used to elucidate the three-dimensional structure of two important TTR variants: TTR Y78F, an amyloidogenic protein, and TTR R104H, which is associated with a protective effect over the amyloidogenic V30M mutation. The structures of those two TTR variants have been determined in space group P21212 to 1.55 and 1.60,Å resolution, respectively, using molecular-replacement techniques. Detailed analysis of the protein model for TTR Y78F indicates a destabilization of the contacts between the ,-helix and AB loop and the body of the molecule, intimately related to the amyloidogenic nature; contrastingly, in the TTR R104H variant new contacts involving the N-terminal region and His104 are clearly antagonists of amyloid formation. [source] Crystallization of a core fragment of the flagellar hook protein FlgEACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Fadel A. Samatey A core fragment of the bacterial flagellar hook protein FlgE was overexpressed, purified and crystallized. The crystal diffracted to 1.6,Å resolution using synchrotron X-radiation. The crystal belongs to the orthorhombic crystal system, with space group P21212 and unit-cell parameters a = 128.4, b = 48.8, c = 96.7,Å. SeMet protein was also overexpressed, purified, crystallized and a set of 2.3,Å MAD data was collected. [source] Crystallization and preliminary X-ray diffraction studies of a fungal hydrolase from Ophiostoma novo-ulmiACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2004Michail N. Isupov Dutch elm disease fungus Ophiostoma novo-ulmi contains a hydrolase activity which catalyses the resolution of racemic ethyl naproxen to the corresponding acid. The recombinant enzyme has been crystallized by the vapour-diffusion method in two crystal forms. The crystals of the first form belong to space group P21212, with unit-cell parameters a = 115.9, b = 174.4, c = 62.1,Å. The enzyme also crystallizes in space group P21212, with unit-cell parameters a = 72.9, b = 212.7, c = 61.7,Å. Synchrotron data have been collected for both crystal forms to 2.6 and 2.3,Å, respectively. A molecular-replacement solution has been found using a remote starting model of a bacterial esterase (23% sequence identity) for both crystal forms. Multicrystal averaging has resulted in interpretable electron-density maps. [source] Expression, purification, crystallization and preliminary crystallographic analysis of phosphoserine aminotransferase from Bacillus alcalophilusACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Anatoly 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 X-ray diffraction studies of a novel alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernixACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2003Jodie E. Guy A novel alcohol dehydrogenase enzyme has been cloned from the hyperthermophilic archaeon Aeropyrum pernix and overexpressed in Escherichia coli. This zinc-containing enzyme has been crystallized by the sitting-drop vapour-diffusion method using PEG 600 as precipitant. The crystals diffract to 1.5,Å resolution and belong to the orthorhombic space group P21212, with unit-cell parameters a = 100.7, b = 103.2, c = 67.5,Å. The asymmetric unit contains two enzyme monomers. Two synchrotron data sets have been collected: one at a wavelength near the absorption edge of zinc and one at a remote wavelength. Three strong zinc-ion positions were visible in the anomalous Patterson map. Two additional weaker zinc ions have been identified by anomalous Fourier synthesis. [source] Purification, crystallization and preliminary structural studies of dTDP-4-keto-6-deoxy-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, the fourth enzyme in the dTDP- l -epivancosamine biosynthetic pathwayACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2002Alexandra B. Merkel The vancomycin class of antibiotics is regarded as the last line of defence against Gram-positive bacteria. The compounds used clinically are very complex organic molecules and are made by fermentation. The biosynthesis of these is complex and fascinating. Its study holds out the prospect of utilizing genetic engineering of the enzymes in the pathway in order to produce novel vancomycin analogues. In part, this requires detailed structural insight into substrate specificity as well as the enzyme mechanism. The crystallization of one of the enzymes in the chloroeremomycin biosynthetic pathway (a member of the vancomycin family), dTDP-3-amino-4-keto 2,3,6-trideoxy-3- C -methyl-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, is reported here. The protein is fourth in the pathway which makes a carbohydrate essential for the activity of chloroeremomycin. The crystals of EvaD diffract to 1.5,Å and have unit-cell parameters a = 98.6, b = 72.0, c = 57.1,Å with space group P21212. Data to this resolution were collected at the European Synchrotron Radiation Facility. [source] Crystallization and preliminary X-ray study of an N-terminal fragment of rat liver ribosomal P2 proteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002David Mandelman Ribosomal P proteins have been shown to be involved in the binding of elongation factors and participate in factor-dependent GTP hydrolysis. The P proteins form the pentamer (P1/P2)2,P0 constituting the lateral flexible stalk of the 60S ribosomal subunit. The highly soluble domain (1,65) of rat liver P2 has been overexpressed in Escherichia coli as an N-terminal poly-His-tagged protein and crystallized. To reduce nucleation and improve crystal morphology and diffraction power, the crystals were grown in a gel matrix and an oil barrier was added between the reservoir and the drop to reduce the rate of vapour diffusion. This dramatically reduced the nucleation in the drops and yielded diffraction-quality crystals. Data were collected to 2.4,Å resolution at beamline ID 14-1, ESRF. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 37.7, b = 96.7, c = 135.0,Å. [source] Mouse testis,brain RNA-binding protein (TB-RBP): expression, purification and crystal X-ray diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001John M. Pascal TB-RBP (testis,brain RNA-binding protein) is a mouse RNA-binding protein that controls the temporal and spatial expression of mRNA. Found most abundantly in brain and male germ cells in the testis, TB-RBP is known to suppress the translation of specific testicular and brain mRNAs as part of cell development. TB-RBP,mRNA complexes can bind microtubules and thereby facilitate RNA translocation. Translin is the human orthologue of TB-RBP which binds to single-stranded ends of DNA sequences in breakpoint regions of chromosomal translocations. TB-RBP/translin has been crystallized in space group P21212. The expression, purification, and crystallization of TB-RBP are described as well as preliminary X-ray diffraction data. The multimeric state of TB-RBP is addressed using dynamic light-scattering results. [source] Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM domainACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2001Keisuke Hamada The Rho guanine nucleotide-dissociation inhibitor (RhoGDI) is a general regulator that forms a complex with the GDP-bound form of Rho-family GTPases and suppresses their activation. The FERM domains of ERM (ezrin/radixin/moesin) proteins bind to RhoGDI and dissociate Rho from RhoGDI. The formation of a complex between RhoGDI and the FERM domain is an important step in the regulatory cycle of Rho activation. In this study, crystals of RhoGDI complexed with the FERM domain of radixin were obtained. The crystals of the binary complex belong to the space group P21212, with unit-cell parameters a = 130.9,(2), b = 151.2,(2), c = 71.2,(1),Å, and contain two protein complexes in the crystallographic asymmetric unit. A 2.9,Å resolution data set was collected using synchrotron radiation at SPring-8. [source] Expression, purification, crystallization and preliminary X-ray analysis of the native class C ,-lactamase from Enterobacter cloacae 908R and two mutantsACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2001J. Wouters Crystals have been obtained of the Enterobacter cloacae 908R ,-lactamase and two point mutants by the vapour-diffusion method using similar conditions [pH 9.0, polyethylene glycol (Mr = 6000) as precipitant]. The three crystal forms belong to the orthorhombic space group P21212, with roughly the same unit-cell parameters; i.e. for the wild-type crystals a = 46.46, b = 82.96, c = 95.31,Å. In the best cases, the crystals diffract to about 2.1,Å resolution on a rotating-anode X-ray source at room temperature. Co-crystallization experiments of poor substrates with the wild-type protein and the active-site serine mutant (S64C) are planned and should lead to a better understanding of the catalytic mechanism of class C ,-lactamases. [source] Crystallization and preliminary crystallographic analysis of N -acetylglucosamine 6-phosphate deacetylase from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2000F. 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] Crystallization and preliminary crystallographic studies of a new crystal form of Escherichia colil -asparaginase II (Ser58Ala mutant)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2000Maciej Kozak Periplasmic Escherichia colil -asparaginase II with an Ser58Ala mutation in the active-site cavity has been crystallized in a new orthorhombic form (space group P21212). Crystals of this polymorph suitable for X-ray diffraction have been obtained by vapour diffusion using two sets of conditions: (i) 1% agarose gel using MPD as precipitant (pH 4.8) and (ii) liquid droplets using PEG-MME 550 (pH 9.0). The crystals grown in agarose gel are characterized by unit-cell parameters a = 226.9, b = 128.4, c = 61.9,Å and diffract to 2.3,Å resolution. The asymmetric unit contains six protein molecules arranged into one pseudo-222-symmetric homotetramer and an active-site competent dimer from which another homotetramer is generated by crystallographic symmetry. [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] Expression, purification and crystallization of Swi5 and the Swi5,Sfr1 complex from fission yeastACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Naoyuki Kuwabara The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (,N180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively. Swi5 was crystallized in two forms belonging to space groups C2 and R3 and the crystals diffracted to 2.7,Å resolution. Swi5,,N180Sfr1 was crystallized in space group P21212 and the crystals diffracted to 2.3,Å resolution. The crystals of Swi5 and Swi5,,N180Sfr1 are likely to contain one tetramer and two heterodimers in the asymmetric unit, respectively. [source] Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-IACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Xenia Bogdanovi DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P21212. The DppA crystal diffracted X-rays to 1.9,Å resolution using an in-house X-ray generator. [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] Crystallization and initial X-ray diffraction studies of the flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from the soil bacterium Paracoccus denitrificansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Klumpler The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8,Å resolution. The crystals of native FerB belonged to space group P21, with unit-cell parameters a = 61.6, b = 110.1, c = 65.2,Å, , = 118.2° and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P21212, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5,Å and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress. [source] Crystallization and preliminary crystallographic analysis of the global nitrogen regulator AmtR from Corynebacterium glutamicumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Kristin Hasselt AmtR, a member of the TetR family of transcription regulators, is a global regulator of nitrogen control in Corynebacterium glutamicum. Unlike other TetR-family members, which are regulated by small-molecule effectors, AmtR is regulated by a protein called GlnK. It has been shown that a GlnK trimer has to become adenylylated prior to formation of a complex with AmtR. The physiological function of AmtR has been very well studied, but structural characterization of the mechanistic aspects of AmtR-regulated transcription has yet to be accomplished. AmtR has successfully been crystallized in space group P21212, with six molecules in the asymmetric unit and unit-cell parameters a = 153.34, b = 163.10, c = 51.93,Å. Preliminary phases were obtained using Se-SAD. [source] Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of ,-ketoacyl-ACP synthase III (FabH) from Xanthomonas oryzae pv. oryzaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Kim-Hung Huynh The bacterial ,-ketoacyl-ACP synthase III (KASIII) encoded by the gene fabH (Xoo4209) from Xanthomonas oryzae pv. oryzae, a plant pathogen, is an important enzyme in the elongation steps of fatty-acid biosynthesis. It is expected to be one of the enzymes responsible for bacterial blight (BB), a serious disease that results in huge production losses of rice. As it represents an important target for the development of new antibacterial drugs against BB, determination of the crystal structure of the KAS III enzyme is essential in order to understand its reaction mechanism. In order to analyze the structure and function of KAS III, the fabH (Xoo4209) gene was cloned and the enzyme was expressed and purified. The KASIII crystal diffracted to 2.05,Å resolution and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 69.8, b = 79.5, c = 62.3,Å. The unit-cell volume of the crystal is compatible with the presence of a single monomer in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.27,Å3,Da,1 and a solvent content of 45.8%. [source] Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragmentACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009William J. McKinstry Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5,Å, and diffracted to 2.0,Å resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone. [source] Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Yong-Fu Li Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1,Å resolution and displayed the symmetry of space group P21. CfaBB exhibited a crystal diffraction limit of 2.3,Å resolution and had the symmetry of space group P21212. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3,Å resolution. These structures were determined using the molecular-replacement method. [source] Expression, purification and crystallization of the cofactor-independent monooxygenase SnoaB from the nogalamycin biosynthetic pathwayACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Hanna Koskiniemi 12-deoxy-nogalonic acid oxygenase (SnoaB) catalyzes the oxygenation of 12-deoxy-nogalonic acid at position 12 to yield nogalonic acid, which is one of the steps in the biosynthesis of the polyketide nogalamycin in Streptomyces nogalater. SnoaB belongs to a family of small cofactor-free oxygenases which carry out oxygenation reactions without the aid of any prosthetic group, cofactor or metal ion. Recombinant SnoaB was crystallized in space group P21212, with unit-cell parameters a = 58.8, b = 114.1, c = 49.5,Å, and these crystals diffracted to 2.4,Å resolution. Recombinant SnoaB does not contain any methionine residues and three double mutants were designed and produced for the preparation of selenomethionine-substituted samples. The selenomethionine-substituted mutant F40M/L89M crystallized in the same space group as the native enzyme. [source] Cloning, expression, crystallization and preliminary X-ray analysis of the first two Ig domains from human Roundabout 1 (Robo1)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2007Cecile Morlot Activation of Roundabout 1 (Robo1) by Slit proteins results in axon repulsion from the midline. Robo1 is a large transmembrane receptor expressed on the axon growth cone and the minimal Robo1-binding region required for Slit activation has been mapped to the N-terminal Ig1,2 domains. The cDNA encoding the first two Ig domains of Robo1 has been cloned and the protein has been expressed in HEK293 EBNA-1 mammalian cells. Here, the purification and crystallization conditions of this Robo1 construct are reported. The crystals are orthorhombic, space group P21212, with unit-cell parameters a = 38.8, b = 69.4, c = 103.3,Å and one molecule in the asymmetric unit. X-ray diffraction data have been collected to 2.8,Å resolution on beamline ID29 at the ESRF. [source] Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2006Makoto Akioka To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8,Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84,Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20,2.1,Å from the MAD data set from the SeMet-substituted crystal were used for phase determination. [source] Monoclinic crystal form of Aspergillus niger,-amylase in complex with maltose at 1.8,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2006A. Vuji Aspergillus niger,-amylase catalyses the hydrolysis of ,-1,4-glucosidic bonds in starch. It shows 100% sequence identity to the A. oryzae homologue (also called TAKA-amylase), three crystal structures of which have been published to date. Two of them belong to the orthorhombic space group P212121 with one molecule per asymmetric unit and one belongs to the monoclinic space group P21 with three molecules per asymmetric unit. Here, the purification, crystallization and structure determination of A. niger,-amylase crystallized in the monoclinic space group P21 with two molecules per asymmetric unit in complex with maltose at 1.8,Å resolution is reported. Furthermore, a novel 1.6,Å resolution orthorhombic crystal form (space group P21212) of the native enzyme is presented. Four maltose molecules are observed in the maltose,,-amylase complex. Three of these occupy active-site subsites ,2 and ,1, +1 and +2 and the hitherto unobserved subsites +4 (Asp233, Gly234) and +5 (Asp235). The fourth maltose molecule binds at the distant binding sites d1 (Tyr382) and d2 (Trp385), also previously unobserved. Furthermore, it is shown that the active-site groove permits different binding modes of sugar units at subsites +1 and +2. This flexibility of the active-site cleft close to the catalytic centre might be needed for a productive binding of substrate chains and/or release of products. [source] | ||||||||||