30.0 (3.18.0) 30,049 (4,672) 6,939 (1,061) 98.7 (97.1) 15.1 (2.38) 7.4 (65.4){ Values in parentheses refer to the highest resolution shell (3.18.0A). 1 Rsym = ShSi |,I.h2Ih,i|/ShSi Ih,i, where ,I.h is the mean intensity for reflection Ih and Ih,i is the intensity of an individual measurement of reflection Ih. The Rfree was calculated using 341 (4.9 ) of the reflections, which were set aside from the refinement. doi:10.1371/journal.pone.0063010.tFigure 2. Structure comparison of apo-XerA and Cre bound to DNA monomers. apo-XerA (grey) and Cre (green) complexed to DNA (gold) are superimposed by their catalytic domains. The XerA catalytic Tyr (Y261) is extruded from the catalytic site. The variable C-terminal aMN helices of XerA are in orange and the C-terminal aMN helices of Cre are in blue. The two N-terminal domains differ in their orientations by about 45u, but exhibit approximately the same concave accessible surface openness. doi:10.1371/journal.pone.0063010.gPLOS ONE | www.plosone.orgStructure of the Archaeal XerA Tyr-RecombinaseFigure 3. State of XerA in solution at low protein concentration in 200 mM NaCl buffer. A. Dots: SAXS experimental data I(q) obtained on the SWING beamline. Red curve: calculated curve from the crystal structure with the missing residues added using BUNCH and SABBAC. B. Crystal structure superimposed on a typical envelope of the protein deduced from the SAXS experimental curve using the program GASBOR. The XerA monomer is in grey with the aMN helices in orange. C. Four models obtained using the program BUNCH from the crystal structure allowing the Nterminal domain to freely rotate. The grey structure is the crystal structure. All other models are in a variation of pink. For all models the agreement between the calculated curve and the experimental one is excellent (x0.80). doi:10.1371/journal.pone.0063010.gbottom strand. Unincorporated nucleotides were removed by spin dialysis. The labeled oligonucleotide was then hybridized with a two-fold excess of unlabeled complementary strand in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). When required, the spacer 59 hydroxyl was phosphorylated by using polynucleotide kinase in the presence of excess unlabeled ATP, prior to hybridization.Pemetrexed disodium buffer.Fludarabine phosphate Electrophoresis was performed for 3 h at 38 V/cm. Product sizes were determined by comparing electrophoretic mobilities of the samples to that of ladders (10 bp DNA step ladder, Promega and 82 oligonucleotide sizing ladder, GE Healthcare).PMID:30125989 Plasmid-based recombination assays were performed as previously described and analysed on 1.2 agarose gels [3].Covalent complex formationReactions were carried out in 20 mL of reaction mixture consisting of 30 mM Tris pH 7.5, 50 mg/mL bovine serum albumin, 50 mM NaCl, 25 nM 59 end-labeled half recombination site and 40 pmol of P. abyssi XerA protein. Reactions were incubated for 2 h at 65uC then quenched in Laemmli loading buffer (final concentrations: 40 mM Tris, pH 6.8, 3 SDS, 8 glycerol, 250 mM b-mercaptoethanol, 0.005 bromophenol blue) and heated for 10 min at 100uC. The reaction products were then analyzed by electrophoresis through a 12 SDS-polyacrylamide gel. Products were visualized by phosphorimaging.Results and Discussion Structure determinationAlthough diffraction-quality crystals of native XerA were obtained, they could not be reproduced when using the SelenoMethionine labeled protein. Therefore the structure of full-length XerA was solved by molecular replacement in two stages,.
