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Hanging-drop Method (hanging-drop + method)
Selected AbstractsGrowth of large protein crystals by a large-scale hanging-drop methodJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2010Keisuke Kakinouchi A method for growing large protein crystals is described. In this method, a cut pipette tip is used to hang large-scale droplets (maximum volume 200,µl) consisting of protein and precipitating agents. A crystal grows at the vapor,liquid interface; thereafter the grown crystal can be retrieved by droplet,droplet contact both for repeated macroseeding and for mounting crystals in a capillary. Crystallization experiments with peroxiredoxin of Aeropyrum pernix K1 (thioredoxin peroxidase, ApTPx) and hen egg white lysozyme demonstrated that this large-scale hanging-drop method could produce a large-volume crystal very effectively. A neutron diffraction experiment confirmed that an ApTPx crystal (6.2,mm3) obtained by this method diffracted to beyond 3.5,Å resolution. [source] Crystallization and preliminary X-ray crystallographic studies on the fungal immunomodulatory protein Fve from the golden needle mushroom (Flammulina velutipes)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2003See Voon Seow The fungal immunomodulatory protein from the edible golden needle mushroom (Flammulina velutipes), designated Fve, is a single polypeptide consisting of 114 amino-acid residues. It is believed to trigger the mitogenic proliferation of T lymphocytes and Th1 cytokine production. Here, it is demonstrated that Fve forms a homodimer in nature. In order to understand the relationship between its structure and function, Fve was crystallized using the hanging-drop method; the protein formed well diffracting crystals within 3,5,d in 2.5% PEG 400, 2.0,M ammonium sulfate and 0.1,M Tris base buffer pH 8.5. The space group of the Fve crystals is either P43212 or P41212, with unit-cell parameters a = b = 96.92, c = 61.42,Å. The crystal contains two molecules per asymmetric unit and diffracts to 1.4,Å resolution when exposed to synchrotron radiation. [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] Preliminary crystallographic study of Thermus aquaticus glycerol kinaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2001Hua-Shan Huang Glycerol kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving glycerol metabolism. GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK -deficient Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94,(3),Å. Native data were collected to 2.87,Å resolution on a Cu,K, rotating-anode X-ray source. [source] Crystallization and preliminary X-ray analyses of catabolite control protein A, free and in complex with its DNA-binding siteACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2000Jan Tebbe The catabolite control protein (CcpA) from Bacillus megaterium is a member of the bacterial repressor protein family GalR/LacI. CcpA with an N-terminal His-tag was used for crystallization. Crystals of free CcpA and of CcpA in complex with the putative operator sequence (catabolite responsive elements, CRE) were obtained by vapour-diffusion techniques at 291,K using the hanging-drop method. CcpA crystals grown in the presence of polyethylene glycol 8000 belong to the hexagonal space group P6122 or P6522, with unit-cell parameters a = 74.4, c = 238.8,Å. These crystals diffract X-rays to 2.55,Å resolution and contain one monomer of the homodimeric protein per asymmetric unit. Crystals of the CcpA,CRE complex were obtained with ammonium sulfate as precipitant and belong to the tetragonal space group I4122, with unit-cell parameters a = 125, c = 400,Å and one complex per asymmetric unit. Although these co-crystals grew to a sufficient size, X-ray diffraction was limited to 8,Å resolution. [source] Crystallization and preliminary X-ray diffraction analysis of a protease-resistant mutant form of human galectin-8ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Hiromi Yoshida A crystal of a protease-resistant mutant form of human galectin-8, a tandem-repeat-type galectin with two carbohydrate-recognition domains, was obtained using the hanging-drop method and was found to belong to the tetragonal space group P43212, with unit-cell parameters a = 78.93, b = 78.93, c = 132.05,Å. Diffraction data were collected to a resolution of 3.4,Å. [source] Expression, purification and preliminary X-ray diffraction studies of VERNALIZATION1208,341 from Arabidopsis thalianaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Gordon King VERNALIZATION1 (VRN1) is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment. Stable suppression of FLC accelerates flowering, a physiological process known as vernalization. VRN1 is a 341-residue DNA-binding protein that contains two plant-specific B3 domains (B3a and B3b), a putative nuclear localization sequence (NLS) and two putative PEST domains. VRN1208,341 includes the second B3 domain and a region upstream that is highly conserved in the VRN1 orthologues of other dicotyledonous plants. VRN1208,341 was crystallized by the hanging-drop method in 0.05,M sodium acetate pH 6.0 containing 1.0,M NaCl and 18%(w/v) PEG 3350. Preliminary X-ray diffraction data analysis revealed that the VRN1208,341 crystal diffracted to 2.1,Å and belonged to space group C2, with unit-cell parameters a = 105.2, b = 47.9, c = 61.2,Å, , = 90.0, , = 115.4, , = 90.0°. Assuming that two molecules occupy the asymmetric unit, a Matthews coefficient of 2.05,Å3,Da,1 and a solvent content of 40.1% were calculated. [source] Purification, crystallization and preliminary crystallographic analysis of the Hermes transposaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2005Zhanita N. Perez DNA transposition is the movement of a defined segment of DNA from one location to another. Although the enzymes that catalyze transposition in bacterial systems have been well characterized, much less is known about the families of transposase enzymes that function in higher organisms. Active transposons have been identified in many insect species, providing tools for gene identification and offering the possibility of altering the genotypes of natural insect populations. One of these active transposons is Hermes, a 2749-base-pair element from Musca domestica that encodes its own transposase. An N-terminally deleted version of the Hermes transposase (residues 79,612) has been overexpressed and purified, and crystals that diffract to 2.1,Å resolution have been obtained at 277,K by the hanging-drop method. [source] Expression, purification and X-ray crystallographic analysis of thioredoxin from Streptomyces coelicolorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2005Petra Stefankova Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disulfide reductases. In turn, they are reduced by electrons obtained from the NADPH-containing thioredoxin reductase. Thioredoxins have been isolated and characterized from a large number of organisms. The Gram-positive bacterium Streptomyces coelicolor contains three thioredoxins that are involved in unknown biological processes. trxA from S. coelicolor was cloned and expressed in Escherichia coli and the protein purified and crystallized using the hanging-drop method of vapour diffusion. The crystal structure of thioredoxin A has been determined at 1.5,Å resolution using a synchrotron-radiation source. The protein reveals a thioredoxin-like fold with a typical CXXC active site. The crystal exhibits the symmetry of space group P21212, with unit-cell parameters a = 43.6, b = 71.8, c = 33.2,Å. [source] | ||||||||||