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Vapor-diffusion Technique (vapor-diffusion + technique)
Selected AbstractsPlasmodium falciparum Rab6 GTPase: expression, purification, crystallization and preliminary crystallographic studiesACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2000Debasish Chattopadhyay The Plasmodium falciparumrab6 gene encodes a 208 amino-acid polypeptide. Two recombinant versions of P. falciparum Rab6 protein were expressed in Escherichia coli: the full-length protein and a truncated form containing residues 1,175. Both forms were purified from the soluble fraction of bacterial extract and were purified by ion-exchange chromatography and size-exclusion chromatography. Purified proteins were crystallized at pH 6.5 using the hanging-drop vapor-diffusion technique at room temperature. The full-length protein diffracted to 2.4,Å and belongs to the tetragonal space group P43212 or P41212, with unit-cell parameters a = b = 80.6, c = 90.4,Å. The crystals of the truncated protein were isomorphous with those of the full-length construct and diffracted X-rays to 2.2,Å resolution. [source] Crystallization of native and selenomethionyl yeast orotidine 5,-phosphate decarboxylaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2000Brian G. Miller Crystals of the Saccharomyces cerevisiae pyrimidine biosynthetic enzyme orotidine 5,-phosphate decarboxylase (ODCase) were grown by the hanging-drop vapor-diffusion technique at 277,K using polyethylene glycol 4000 as the precipitant. Crystals of native and selenomethionyl ODCase diffract to less than 2.2,Å and belong to the orthorhombic space group P212121, with unit-cell parameters a = 90.1, b = 116.2, c = 117.0,Å. Crystals of ODCase grown in the presence of the postulated transition-state analog inhibitor 6-hydroxyuridine 5,-phosphate (BMP) diffract to less than 2.5,Å and belong to space group P21, with unit-cell parameters a = 79.9, b = 80.0, c = 98.2,Å, , = 108.6°. [source] Crystallization of human complement component C3b in the presence of a staphylococcal complement-inhibitor protein (SCIN)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Brandon L. Garcia Staphylococcus aureus secretes a number of small proteins that effectively attenuate the human innate immune response. Among these, the staphylococcal complement-inhibitor protein (SCIN) disrupts the function of the complement component 3 (C3) convertase that is initiated through either the classical or the alternative pathway and thereby prevents amplification of the complement response on the bacterial surface. Recent studies have shown that SCIN may affect the activities of the C3 convertase by binding in an equimolar fashion to C3b, which is itself an integral although non-enzymatic component of the convertase. In order to better understand the nature of the C3b,SCIN interaction, the hanging-drop vapor-diffusion technique was used to crystallize human C3b in the presence of a recombinant form of SCIN. These crystals diffracted synchrotron X-rays to approximately 6,Å Bragg spacing and grew in a primitive tetragonal space group (P41212 or P43212; unit-cell parameters a = b = 128.03, c = 468.59,Å). Cell-content analysis of these crystals was consistent with the presence of either two 1:1 complexes or a single 2:2 assembly in the asymmetric unit, both of which correspond to a solvent content of 51.9%. By making use of these crystals, solution of the C3b,SCIN structure should further our understanding of complement inhibition and immune evasion by this pathogen. [source] Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2006Marina N. Vassylyeva The bacterial transcriptional factor and virulence regulator RfaH binds to rapidly moving transcription elongation complexes through specific interactions with the exposed segment of the non-template DNA strand. To elucidate this unusual mechanism of recruitment, determination of the three-dimensional structure of RfaH and its complex with DNA was initiated. To this end, the Escherichia coli rfaH gene was cloned and expressed. The purified protein was crystallized by the sitting-drop vapor-diffusion technique. The space group was P6122 or P6522, with unit-cell parameters a = b = 45.46, c = 599.93,Å. A complex of RfaH and a nine-nucleotide oligodeoxyribonucleotide was crystallized by the same technique, but under different crystallization conditions, yielding crystals that belonged to space group P1 (unit-cell parameters a = 36.79, b = 44.01, c = 62.37,Å, , = 80.62, , = 75.37, , = 75.41°). Complete diffraction data sets were collected for RfaH and its complex with DNA at 2.4 and 1.6,Å resolution, respectively. Crystals of selenomethionine-labeled proteins in both crystal forms were obtained by cross-microseeding using the native microcrystals. The structure determination of RfaH and its complex with DNA is in progress. [source] Cloning, expression, purification, crystallization and initial crystallographic analysis of transcription elongation factors GreB from Escherichia coli and Gfh1 from Thermus thermophilusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2006Anna A. Perederina The Escherichia coli gene encoding the transcription cleavage factor GreB and the Thermus thermophilus gene encoding the anti-GreA transcription factor Gfh1 were cloned and expressed and the purified proteins were crystallized by the sitting-drop vapor-diffusion technique. The GreB and Gfh1 crystals, which were improved by macroseeding, belong to space group P41212 (or P43212), with unit-cell parameters a = b = 148, c = 115.2,Å and a = b = 59.3, c = 218.9,Å, respectively. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8,Å resolution, respectively. Crystals of the selenomethionine proteins were obtained by microseeding using the native protein crystals and diffract as well as the native ones. The structure determination of these proteins is now in progress. [source] | ||||||||||