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Rfree Factors (rfree + factor)
Selected AbstractsDe novo sulfur SAD phasing of the lysosomal 66.3,kDa protein from mouseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Kristina Lakomek The 66.3,kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75,kDa preproprotein which is further processed into 28 and 40,kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3,kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3,kDa variant and the double-chain form consisting of a 28,kDa fragment and a 40,kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3,kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9,Å on the BESSY beamline BL14.2 of Freie Universität Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4,Å using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an Rfree factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks. [source] Structure of photosynthetic glyceraldehyde-3-phosphate dehydrogenase (isoform A4) from Arabidopsis thaliana in complex with NADACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Simona Fermani The crystal structure of the A4 isoform of photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana, expressed in recombinant form and complexed with NAD, is reported. The crystals, which were grown in 2.4,M ammonium sulfate and 0.1,M sodium citrate, belonged to space group I222. The asymmetric unit includes ten subunits, i.e. two independent tetramers plus a dimer that generates a third tetramer by a crystallographic symmetry operation. The crystal structure was solved by molecular replacement and refined to an R factor of 23.7% and an Rfree factor of 28.9% at 2.6,Å resolution. In the final model, each subunit binds one NAD+ molecule and two sulfates, which occupy the Ps and the Pi anion-binding sites. Detailed knowledge of this structure is instrumental for structural investigation of supramolecular complexes of A4 -GAPDH, phosphoribulokinase and CP12, which are involved in the regulation of photosynthesis in the model plant A. thaliana. [source] On the use of logarithmic scales for analysis of diffraction dataACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Alexandre Urzhumtsev Predictions of the possible model parameterization and of the values of model characteristics such as R factors are important for macromolecular refinement and validation protocols. One of the key parameters defining these and other values is the resolution of the experimentally measured diffraction data. The higher the resolution, the larger the number of diffraction data Nref, the larger its ratio to the number Nat of non-H atoms, the more parameters per atom can be used for modelling and the more precise and detailed a model can be obtained. The ratio Nref/Nat was calculated for models deposited in the Protein Data Bank as a function of the resolution at which the structures were reported. The most frequent values for this distribution depend essentially linearly on resolution when the latter is expressed on a uniform logarithmic scale. This defines simple analytic formulae for the typical Matthews coefficient and for the typically allowed number of parameters per atom for crystals diffracting to a given resolution. This simple dependence makes it possible in many cases to estimate the expected resolution of the experimental data for a crystal with a given Matthews coefficient. When expressed using the same logarithmic scale, the most frequent values for R and Rfree factors and for their difference are also essentially linear across a large resolution range. The minimal R -factor values are practically constant at resolutions better than 3,Å, below which they begin to grow sharply. This simple dependence on the resolution allows the prediction of expected R -factor values for unknown structures and may be used to guide model refinement and validation. [source] Structure of the transcription regulator CcpA from Lactococcus lactisACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2007Bernhard Loll Catabolite control protein A (CcpA) functions as master transcriptional regulator of carbon catabolism in Firmicutes. It belongs to the family of bacterial repressor/regulator proteins. Here, the crystal structure of the 76,kDa homodimeric CcpA protein from Lactococcus lactis subsp. lactis IL1403 is presented at 1.9,Å resolution in the absence of cognate DNA. The phases were derived by molecular replacement and the structure was refined to crystallographic R and Rfree factors of 0.177 and 0.211, respectively. The presence of a sulfate molecule in the direct vicinity of a putative effector-binding site in the monomer allowed the derivation of a model for the possible binding of small organic effector molecules. [source] Crystal structure reveals two alternative conformations in the active site of ribonuclease Sa2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2004Jozef Three different strains of Streptomyces aureofaciens produce the homologous ribonucleases Sa, Sa2 and Sa3. The crystal structures of ribonuclease Sa (RNase Sa) and its complexes with mononucleotides have previously been reported at high resolution. Here, the structures of two crystal forms (I and II) of ribonuclease Sa2 (RNase Sa2) are presented at 1.8 and 1.5 Å resolution. The structures were determined by molecular replacement using the coordinates of RNase Sa as a search model and were refined to R factors of 17.5 and 15.0% and Rfree factors of 21.8 and 17.2%, respectively. The asymmetric unit of crystal form I contains three enzyme molecules, two of which have similar structures to those seen for ribonuclease Sa, with Tyr87 at the bottom of their active sites. In the third molecule, Tyr87 has moved substantially: the CA atom moves almost 5,Å and the OH of the side chain moves 10,Å, inserting itself into the active site of a neighbouring molecule at a similar position to that observed for the nucleotide base in RNase Sa complexes. The asymmetric unit of crystal form II contains two Sa2 molecules, both of which are similar to the usual Sa structures. In one molecule, two main-chain conformations were modelled in the ,-helix. Finally, a brief comparison is made between the conformations of the Sa2 molecules and those of 34 independent molecules taken from 20 structures of ribonuclease Sa and two independent molecules taken from two structures of ribonuclease Sa3 in various crystal forms. [source] Structure of the catalytic domain of Streptococcus pneumoniae sialidase NanAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2008Guogang Xu Streptococcus pneumoniae genomes encode three sialidases, NanA, NanB and NanC, which are key virulence factors that remove sialic acids from various glycoconjugates. The enzymes have potential as drug targets and also as vaccine candidates. The 115,kDa NanA is the largest of the three sialidases and is anchored to the bacterial membrane. Although recombinantly expressed full-length NanA was soluble, it failed to crystallize; therefore, a 56.5,kDa domain that retained full enzyme activity was subcloned. The purified enzyme was crystallized in 0.1,M MES pH 6.5, 30%(w/v) PEG 4000 using the sitting-drop vapour-diffusion method. Data were collected at 100,K to 2.5,Å resolution from a crystal grown in the presence of the inhibitor 2-deoxy-2,3-dehydro- N -acetyl neuraminic acid. The crystal belongs to space group P212121, with unit-cell parameters a = 49.2, b = 95.6, c = 226.6,Å. The structure was solved by molecular replacement and refined to final R and Rfree factors of 0.246 and 0.298, respectively. [source] | ||||||||||