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Hanging-drop Vapor-diffusion Method (hanging-drop + vapor-diffusion_method)
Selected AbstractsPurification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigenACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004Shuh-Chyung Song The NNA7 Fab fragment recognizes the human glycopeptide N blood-group antigen and has a high affinity for N-type glycophorin A (GPA). To provide insight into how antibodies recognize glycopeptide antigens, soluble Fab fragments were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method at 293,K. The best crystals were obtained from solutions of PEG monomethyl ether 5000 containing 4,8,mM yttrium chloride (YCl3). This rare-earth ion, which could be substituted with various lanthanides, changed the habit of crystals from multinucleated rods with a diffraction limit of 4.25,Å resolution to a diamond-shaped morphology that grew as single crystals and diffracted X-rays to 1.75,Å resolution. Data were collected that indicated that the crystals belonged to space group P212121, with unit-cell parameters a = 57.9, b = 77.1, c = 118.1,Å and one Fab fragment per asymmetric unit. A molecular-replacement solution has been obtained and 86% of the molecule was fitted by use of an automated refinement procedure (ARP). [source] Crystallization and preliminary X-ray crystallographic studies of recombinant human betaine,homocysteine S-methyltransferaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2001Nandita Bose Betaine,homocysteine S-methyltransferase (BHMT) catalyzes a reaction essential for regulation of methionine and homocysteine metabolism and the catabolism of choline in mammalian tissues. Human recombinant BHMT (MW = 45,kDa) has been crystallized by the hanging-drop vapor-diffusion method at 294,K using ethylene glycol as the precipitant. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 109.190, b = 91.319, c = 88.661,Å, , = 122.044°, and diffract to 2.9,Å resolution on a local rotating-anode X-ray source. Rotation-function analysis and the Matthews coefficient, VM = 2.46,Å3,Da,1, are consistent with a dimer in the asymmetric unit, suggesting that the active enzyme is a tetramer with 222 symmetry. [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 crystallographic analysis of the ArsM arsenic(III) S -adenosylmethionine methyltransferaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Kavitha Marapakala Arsenic is the most ubiquitous environmental toxin and carcinogen and consequently ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. A common biotransformation is methylation to monomethylated, dimethylated and trimethylated species. Methylation is catalyzed by the ArsM (or AS3MT) arsenic(III) S -adenosylmethionine methyltransferase, an enzyme (EC 2.1.1.137) that is found in members of every kingdom from bacteria to humans. ArsM from the thermophilic alga Cyanidioschyzon sp. 5508 was expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 84.85, b = 46.89, c = 100.35,Å, , = 114.25° and one molecule in the asymmetric unit. Diffraction data were collected at the Advanced Light Source and were processed to a resolution of 1.76,Å. [source] Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgXACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Joel T. Weadge AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a = 46.4, b = 120.6, c = 86.9,Å, , = 95.7°. The crystals exhibited the symmetry of space group P21 and diffracted to a minimum d -spacing of 2.1,Å. On the basis of the Matthews coefficient (VM = 2.25,Å3,Da,1), two molecules were estimated to be present in the asymmetric unit. [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 crystal-packing studies of Chlorella virus deoxyuridine triphosphataseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Kohei Homma The 141-amino-acid deoxyuridine triphosphatase (dUTPase) from the algal Chlorella virus IL-3A and its Glu81Ser/Thr84Arg-mutant derivative Mu-22 were crystallized using the hanging-drop vapor-diffusion method at 298,K with polyethylene glycol as the precipitant. An apo IL-3A dUTPase with an amino-terminal T7 epitope tag and a carboxy-terminal histidine tag yielded cubic P213 crystals with unit-cell parameter a = 106.65,Å. In the presence of dUDP, the enzyme produced thin stacked orthorhombic P222 crystals with unit-cell parameters a = 81.0, b = 96.2, c = 132.8,Å. T7-histidine-tagged Mu-22 dUTPase formed thin stacked rectangular crystals. Amino-terminal histidine-tagged dUTPases did not crystallize but formed aggregates. Glycyl-seryl-tagged dUTPases yielded cubic P213 IL-3A crystals with unit-cell parameter a = 105.68,Å and hexagonal P63 Mu-22 crystals with unit-cell parameters a = 132.07, c = 53.45,Å, , = 120°. Owing to the Thr84Arg mutation, Mu-22 dUTPase had different monomer-to-monomer interactions to those of IL-3A dUTPase. [source] Expression, refolding, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgEACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009John C. C. Whitney AlgE is an outer membrane protein present in alginate-producing (mucoid) Pseudomonas aeruginosa. AlgE has been overexpressed in insoluble inclusion bodies, purified under denaturing conditions and refolded in a buffer containing decyl ,- d -maltopyranoside. Purified refolded AlgE was detergent-exchanged into n -octyl tetraoxyethylene and diffraction-quality crystals were grown using the hanging-drop vapor-diffusion method. The crystals grew as small hexagons with unit-cell parameters a = 98.8, b = 156.8, c = 90.4,Å, , = , = , = 90.0°. The crystals exhibited the symmetry of space group C222 and diffracted to a minimum d -spacing of 3.0,Å. On the basis of the Matthews coefficient (VM = 3.28,Å3,Da,1), one molecule is estimated to be present in the asymmetric unit. [source] Expression, purification, crystallization and preliminary X-ray studies of the Ebola VP35 interferon inhibitory domainACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Daisy W. Leung Ebola VP35 is a multifunctional protein that is important for host immune suppression and pathogenesis. VP35 contains an N-terminal oligomerization domain and a C-terminal interferon inhibitory domain (IID). Mutations within the VP35 IID result in loss of host immune suppression. Here, efforts to crystallize recombinantly overexpressed VP35 IID that was purified from Escherichia coli are described. Native and selenomethionine-labeled crystals belonging to the orthorhombic space group P212121 were obtained by the hanging-drop vapor-diffusion method and diffraction data were collected at the ALS synchrotron. [source] Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis,/,-type small acid-soluble spore protein and DNAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2007Daniela Bumbaca An engineered variant of an ,/,-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281,K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4,Å resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6122 or P6522, with unit-cell parameters a = b = 87.0, c = 145.4,Å, , = , = 90.0, , = 120.0°. Diffraction data were 96.6% complete to 2.4,Å resolution, with an Rsym of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress. [source] | ||||||||||