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Hanging-drop Technique (hanging-drop + technique)
Selected AbstractsHeavy-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] Structure of mouse L-chain ferritin at 1.6,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001Thierry Granier Cubic F432 crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique with ammonium sulfate and cadmium sulfate as precipitants. The structure was refined to 2.1 and 1.6,Å resolution from data obtained at room temperature and under cryogenic conditions, respectively. The structure of an eight-amino-acid loop insertion in the mouse sequence is found to be highly disordered both at room temperature and at low temperature. [source] Crystallization and preliminary X-ray diffraction data of mouse L-chain apoferritin crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2000Thierry Granier Crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique using ammonium sulfate as precipitant. Two crystal forms were observed in the same drop. The crystals belong to either the P2 monoclinic or to the P4212 tetragonal space group. The monoclinic crystals diffracted to beyond 2.4,Å resolution but were systematically twinned, while the tetragonal crystals diffracted to beyond 2.9,Å. These crystallization conditions in the absence of metal salts should facilitate the study of the interaction between L-chain ferritins and heavy metals, particularly the iron core. [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 X-ray characterization of the Skp1,Fbg3 complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Taichi Kumanomidou F-box proteins are the substrate-recognition components of Skp1,Cullin1,F-box protein,Rbx1 (SCF) ubiquitin ligase complexes. Fbs1, an F-box protein, binds specifically to proteins modified with high-mannose oligosaccharides. Fbg3, another F-box protein, has 51% sequence identity to Fbs1. Although the residues that are necessary for binding to oligosaccharides are conserved between Fbs1 and Fbg3, Fbg3 does not bind glycoproteins. Skp1 and Fbg3 were co-expressed in Escherichia coli and their complex was purified to homogeneity and crystallized. Microseeding combined with the sandwiched hanging-drop technique improved the quality of the resulting crystals. The plate-shaped crystals belonged to space group P212121, with unit-cell parameters a = 34.1, b = 76.6, c = 193.9,Å and one molecule per asymmetric unit. [source] Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2aACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2005Wenxin Hu Diadenosine tetraphosphate (Ap4A) hydrolase (EC 3.6.1.41) hydrolyzes Ap4A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap4A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3,kDa Ap4A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap4A hydrolase have been obtained by the hanging-drop technique at 291,K using PEG 550 MME as precipitant. Ap4A hydrolase crystals diffract X-rays to 3.26,Å and belong to space group P21, with unit-cell parameters a = 118.9, b = 54.6, c = 128.5,Å, , = 95.7°. [source] | ||||||||||