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Rotating-anode Generator (rotating-anode + generator)
Selected AbstractsCharacterization of gadolinium complexes for SAD phasing in macromolecular crystallography: application to CbpFACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Rafael Molina Seven Gd complexes were used in the preparation of heavy-atom derivatives for solving the structure of choline-binding protein F (CbpF), a 36,kDa surface protein from Streptococcus pneumoniae, by the SAD method. CbpF was used as a model system to analyse the phasing capability of each of the derivatives. Three different aspects have been systematically characterized: the efficacy of cocrystallization versus soaking in the binding of the different Gd complexes, their mode of interaction and a comparative study of SAD phasing using synchrotron radiation and using a rotating-anode generator. This study reveals the striking potential of these complexes for SAD phasing using a laboratory source and further reinforces their relevance for high-throughput macromolecular crystallography. [source] The structure of Vibrio cholerae extracellular endonuclease I reveals the presence of a buried chloride ionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2006Bjørn Altermark The crystal structure of a periplasmic/extracellular endonuclease from Vibrio cholerae has been solved at low and at neutral pH. Crystals grown at pH 4.6 and 6.9 diffracted to 1.6,Å (on BM01A at the ESRF) and 1.95,Å (on a rotating-anode generator), respectively. The structures of the endonuclease were compared with the structure of a homologous enzyme in V. vulnificus. The structures of the V. cholerae enzyme at different pH values are essentially identical to each other and to the V. vulnificus enzyme. However, interesting features were observed in the solvent structures. Both V. cholerae structures reveal the presence of a chloride ion completely buried within the core of the protein, with the nearest solvent molecule approximately 7,Å away. Magnesium, which is essential for catalysis, is present in the structure at neutral pH, but is absent at low pH, and may partly explain the inactivity of the enzyme at lower pH. [source] Crystallization and preliminary X-ray diffraction analysis of homing endonuclease I- Tsp061IACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Takahito Imagawa Two crystal forms, rhombohedral and hexagonal, of a homing endonuclease from Thermoproteus sp. IC-061 (I- Tsp0611) were obtained by the hanging-drop and sitting-drop method, respectively. The hexagonal crystals belong to space group P6322, with unit-cell parameters a = b = 111.4, c = 97.6,Å, and diffract to 3.2,Å resolution on beamline BL44 at SPring-8 (Harima, Japan). The rhombohedral crystals belong to space group R32, with unit-cell parameters a = b = 95.4, c = 192.9,Å, and diffract to 2.7,Å resolution using a Cu,K, rotating-anode generator with an R-AXIS VII detector. The crystal asymmetric unit contained one protein molecule and the solvent contents of the two crystal forms were estimated to be 68.3 and 67.6% by volume, respectively. [source] Heavy-atom derivatives in lipidic cubic phases: results on hen egg-white lysozyme tetragonal derivative crystals with Gd-HPDO3A complexACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004Éric Girard Gd-HPDO3A, a neutral gadolinium complex, is a good candidate for obtaining heavy-atom-derivative crystals by the lipidic cubic phase crystallization method known to be effective for membrane proteins. Gadolinium-derivative crystals of hen egg-white lysozyme were obtained by co-crystallizing the protein with 100,mM Gd-HPDO3A in a monoolein cubic phase. Diffraction data were collected to a resolution of 1.7 Å using Cu,K, radiation from a rotating-anode generator. Two binding sites of the gadolinium complex were located from the strong gadolinium anomalous signal. The Gd-atom positions and their refined occupancies were found to be identical to those found in derivative crystals of hen egg-white lysozyme obtained by co-crystallizing the protein with 100,mM Gd-HPDO3A using the hanging-drop technique. Moreover, the refined structures are isomorphous. The lipidic cubic phase is not disturbed by the high concentration of Gd-HPDO3A. This experiment demonstrates that a gadolinium complex, Gd-HPDO3A, can be used to obtain derivative crystals by the lipidic cubic phase crystallization method. Further studies with membrane proteins that are known to crystallize in lipidic cubic phases will be undertaken with Gd-HPDO3A and other Gd complexes to test whether derivative crystals with high Gd-site occupancies can be obtained. [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 diffraction studies of a novel alkaline serine protease (KP-43) from alkaliphilic Bacillus sp. strain KSM-KP43ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2001Tsuyoshi Nonaka A novel alkaline serine protease (KP-43) which belongs to a new class of the subtilisin superfamily was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. The crystals belong to the orthorhombic space group C2221, with unit-cell parameters a = 43.50,(2), b = 110.4,(1), c = 168.9,(1),Å. The crystals diffract X-rays beyond 1.9,Å resolution using Cu,K, radiation from a rotating-anode generator and are suitable for high-resolution crystal structure analysis. [source] Crystallization and X-ray diffraction studies of the fatty-acid responsive transcription factor FadR from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2000Daan M. F. Van Aalten FadR, an acylCoA-dependent Escherichia coli transcription factor controlling the expression of genes involved in fatty-acid degradation and synthesis, has been crystallized. Crystals of two binary complexes were obtained. The FadR,CoA complex crystallized in space group C2221, with unit-cell parameters a = 61.3, b = 102.0, c = 91.3,Å. The FadR,octanoyl-CoA complex crystallized in space group P6522, with unit-cell parameters a = b = 59.7, c = 296.2,Å. Both crystal forms diffracted to 3.5,Å on a rotating-anode generator. In both crystal forms, the asymmetric unit contains one subunit. The protein is known to be a homodimer; each subunit consists of two domains of unknown fold. For the acyl-CoA-binding domain, a previously undetected sequence homology to PAS domains, in particular the photoactive yellow protein, is reported. [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] Crystallization of the pneumococcal autolysin LytC: in-house phasing using novel lanthanide complexesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Inmaculada Pérez-Dorado LytC, one of the major autolysins from the human pathogen Streptococcus pneumoniae, has been crystallized as needles by the hanging-drop technique using 10%(w/v) PEG 3350 as precipitant and 10,mM HEPES pH 7.5. LytC crystals were quickly soaked in mother liquor containing 2,mM of the complex Gd-HPDO3A to produce derivatized crystals (LytCGd-HPDO3A). Both native LytC and isomorphous LytCGd-HPDO3A crystals were flash-cooled in a nitrogen flow at 120,K prior to X-ray data collection using an in-house Enraf,Nonius rotating-anode generator (, = 1.5418,Å) and a MAR345 imaging-plate detector. In both cases, good-quality diffraction patterns were obtained at high resolution. LytCGd-HPDO3A crystals allowed the collection of a SAD X-ray data set to 2.6,Å resolution indexed in terms of a P21 monoclinic unit cell with parameters a = 59.37, b = 67.16, c = 78.85,Å, , = 105.69°. The anomalous Patterson map allowed the identification of one heavy-atom binding site, which was sufficient for the calculation of an interpretable anomalous map at 2.6,Å resolution. [source] Crystallization and preliminary diffraction analysis of a ,-galactosidase from Trichoderma reeseiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Mirko Maksimainen An extracellular ,-galactosidase from Trichoderma reesei was crystallized from sodium cacodylate buffer using polyethylene glycol (PEG) as a precipant. Crystals grown by homogenous streak-seeding belonged to space group P1, with unit-cell parameters a = 67.3, b = 69.1, c = 81.5,Å, , = 109.1, , = 97.3, , = 114.5°. The crystals diffracted to 1.8,Å resolution using a rotating-anode generator and to 1.2,Å resolution using a synchrotron source. On the basis of the Matthews coefficient (VM = 3.16,Å3,Da,1), one molecule is estimated to be present in the asymmetric unit. The aim of the determination of the crystal structure is to increase the understanding of this industrially significant enzyme. [source] Crystallization and preliminary X-ray diffraction analysis of the , subunit Yke2 of the Gim complex from Saccharomyces cerevisiaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2008Rebeca Pérez de Diego The Gim complex (GimC) from Saccharomyces cerevisiae is a heterohexameric protein complex, also known as prefoldin (PFD), which binds and stabilizes unfolded target polypeptides and subsequently delivers them to chaperonins for completion of folding. In this study, the crystallization and preliminary X-ray analysis of one of the , subunits of the Gim complex (Yke2) from S. cerevisiae are described. The purified protein was crystallized by the vapour-diffusion method, producing two types of crystals that belonged to the orthorhombic space group C222 or the primitive monoclinic space group P21. The unit-cell parameters for the C -centred orthorhombic crystal were a = 48.2, b = 168.86, c = 131.81,Å and the unit-cell parameters for the primitive monoclinic crystal were a = 47.83, b = 134.90, c = 81.50,Å, , = 100.71°. The Yke2 crystals diffracted to 4.2 and 3.1,Å resolution, respectively, on a rotating-anode generator under cryoconditions. This is the first report concerning the crystallization of a , subunit of a eukaryotic prefoldin. [source] Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2006Francisca Sevilla A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6,kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8,Å from a single crystal flash-cooled at 100,K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23,Å, , = 102.90, , = 104.40, , = 99.07°, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress. [source] Purification, crystallization and preliminary X-ray diffraction analysis of pathogen-inducible oxygenase (PIOX) from Oryza sativaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2006Tracy Lloyd Pathogen-inducible oxygenase (PIOX) is a heme-containing membrane-associated protein found in monocotyledon and dicotyledon plants that utilizes molecular oxygen to convert polyunsaturated fatty acids into their corresponding 2R -hydroperoxides. PIOX is a member of a larger family of fatty-acid ,-dioxygenases that includes the mammalian cyclooxygenase enzymes cyclooxygenase 1 and 2 (COX-1 and COX-2). Single crystals of PIOX from rice (Oryza sativa) have been grown from MPD using recombinant protein expressed in Escherichia coli and subsequently extracted utilizing decyl maltoside as the solubilizing detergent. Crystals diffract to 3.0,Å resolution using a rotating-anode generator and R-AXIS IV detector, and belong to space group P1. Based on the Matthews coefficient and self-rotation function analyses, there are presumed to be four molecules in the asymmetric unit related by noncrystallographic 222 symmetry. [source] Recombinant bovine uteroglobin at 1.6,Å resolution: a preliminary X-ray crystallographic analysisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2005Victoria Von Der Decken Uteroglobin (UG) is a conserved protein which is induced by progesterone and secreted by the epithelia of various mammalian reproductive and respiratory organs. Recombinant bovine uteroglobin (recbUG), consisting of 80 amino acids with a C-terminal His6 tag, was overexpressed in Escherichia coli and purified. The protein was crystallized in two geometric forms, rhomboid and cuneate (wedge-shaped), by the hanging-drop vapour-diffusion method at 295,K. The rhomboid crystals diffracted to a maximum resolution of 1.6,Å using synchrotron radiation. These crystals belong to space group P21212, with unit-cell parameters a = 81.42, b = 82.82, c = 45.26,Å, and contain four monomers per asymmetric unit. The cuneate crystals diffracted to 2.35,Å resolution using a rotating-anode generator. These crystals belong to space group C2221, with unit-cell parameters a = 43.39, b = 93.94, c = 77.30,Å, and contain two molecules per asymmetric unit. [source] Refolding, crystallization and preliminary X-ray structural studies of the West Nile virus envelope (E) protein domain IIIACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2005Zhiyong Lou Domain III of the West Nile virus envelope protein, the putative receptor-binding domain, is a major virion-surface determinant for virulence. This protein was reported to be intrinsically unstable and has defied previous crystallization attempts. It has now been purified from inclusion bodies by protein refolding and was crystallized using the hanging-drop vapour-diffusion method at 291,K. The crystals belong to space group P2221, with unit-cell parameters a = 52.6, b = 59.7, c = 95.0,Å. A complete data set was collected to 2.8,Å at 100,K with Cu,K, X-rays from a rotating-anode generator. [source] Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage ,29ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2005Dwight L. Anderson The Bacillus subtilis bacteriophage ,29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293,K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P41212, with unit-cell parameters a = b = 77.13, c = 37.12,Å. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34,Å. Complete data sets have been collected to 1.78 and 1.80,Å for forms I and II, respectively, at 100,K using Cu,K, X-rays from a rotating-anode generator. Calculation of a VM value of 2.46,Å3,Da,1 for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a VM of 4.80,Å3,Da,1 with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7. [source] | ||||||||||