Home About us Contact
|
Home About us Contact | ||
|
|
||
Molecular-replacement Techniques (molecular-replacement + techniques)
Selected AbstractsX-ray crystallographic studies of two transthyretin variants: further insights into amyloidogenesisACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2005Ricardo M. Neto-Silva Transthyretin (TTR) is a homotetrameric plasma protein that, as a result of a set of not yet fully characterized conformational changes, forms fibrillar aggregates that are the major protein component of amyloid deposits. More than 80 mutations associated with TTR amyloid deposition have been described in the literature. X-ray crystallography was used to elucidate the three-dimensional structure of two important TTR variants: TTR Y78F, an amyloidogenic protein, and TTR R104H, which is associated with a protective effect over the amyloidogenic V30M mutation. The structures of those two TTR variants have been determined in space group P21212 to 1.55 and 1.60,Å resolution, respectively, using molecular-replacement techniques. Detailed analysis of the protein model for TTR Y78F indicates a destabilization of the contacts between the ,-helix and AB loop and the body of the molecule, intimately related to the amyloidogenic nature; contrastingly, in the TTR R104H variant new contacts involving the N-terminal region and His104 are clearly antagonists of amyloid formation. [source] Crystallization and preliminary crystallographic analysis of endonuclease VIII in its uncomplexed formACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004Gali Golan The Escherichia coli DNA repair enzyme endonuclease VIII (EndoVIII or Nei) excises oxidized pyrimidines from damaged DNA substrates. It overlaps in substrate specificity with endonuclease III and may serve as a back-up for this enzyme in E. coli. The three-dimensional structure of Nei covalently complexed with DNA has been recently determined, revealing the critical amino-acid residues required for DNA binding and catalytic activity. Based on this information, several site-specific mutants of the enzyme have been tested for activity against various substrates. Although the crystal structure of the DNA-bound enzyme has been fully determined, the important structure of the free enzyme has not previously been analyzed. In this report, the crystallization and preliminary crystallographic characterization of DNA-free Nei are described. Four different crystal habits are reported for wild-type Nei and two of its catalytic mutants. Despite being crystallized under different conditions, all habits belong to the same crystal form, with the same space group (I222) and a similar crystallographic unit cell (average parameters a = 57.7, b = 80.2, c = 169.7,Å). Two of these crystal habits, I and IV, appear to be suitable for full crystallographic analysis. Crystal habit I was obtained by vapour diffusion using PEG 8000, glycerol and calcium acetate. Crystal habit IV was obtained by a similar method using PEG 400 and magnesium chloride. Both crystals are mechanically strong and stable in the X-ray beam once frozen under cold nitrogen gas. A full diffraction data set has recently been collected from a wild-type Nei crystal of habit I (2.6,Å resolution, 85.2% completeness, Rmerge = 9.8%). Additional diffraction data were collected from an Nei-R252A crystal of habit IV (2.05,Å resolution, 99.9% completeness, Rmerge = 6.0%) and an Nei-E2A crystal of habit IV (2.25,Å resolution, 91.7% completeness, Rmerge = 6.2%). These diffraction data were collected at 95,100 K using a synchrotron X-ray source and a CCD area detector. All three data sets are currently being used to obtain crystallographic phasing via molecular-replacement techniques. [source] Structure of pteridine reductase (PTR1) from Leishmania tarentolaeACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2003Haiyan Zhao The protozoan parasites Leishmania utilize a pteridine-reducing enzyme, pteridine reductase (PTR1), to bypass antifolate inhibition. The crystal structure of PTR1 from L. tarentolae has been solved as a binary complex with NADPH at 2.8,Å resolution. The structure was solved by molecular-replacement techniques using the recently reported L. major PTR1 structure as a search model. Comparisons of the present structure with the L. major PTR1 allowed us to identify regions of flexibility in the molecule. PTR1 is a member of the growing family of short-chain dehydrogenases (SDR) which share the characteristic Tyr(Xaa)3Lys motif in the vicinity of the active site. The functional enzyme is a tetramer and the crystallographic asymmetric unit contains a tetramer with 222 point-group symmetry. [source] Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with rosmarinic acidACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Juliana I. Dos Santos PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A2 from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8,Å resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P212121, indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A2 structures belong to space group P3121, while the crystals of complexed structures belong to space groups P21 or P212121. [source] Crystallization and preliminary X-ray analysis of ,C,S lyases from two oral streptococciACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Yuichiro Kezuka Hydrogen sulfide, which causes oral malodour, is generally produced from l -cysteine by the action of ,C,S lyase from oral bacteria. The ,C,S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, have been cloned, overproduced, purified and crystallized. X-ray diffraction data were collected from the two types of crystals using synchrotron radiation. The crystal of S. anginosus,C,S lyase belonged to the orthorhombic space group P212121, with unit-cell parameters a = 67.0, b = 111.1, c = 216.4,Å, and the crystal of S. gordonii,C,S lyase belonged to the same space group, with unit-cell parameters a = 58.0, b = 73.9. c = 187.6,Å. The structures of the ,C,S lyases were solved by molecular-replacement techniques. [source] | ||||||||||