title: Crystallization and X-ray diffraction of an ectoine hydroxylase from Bacillus pseudofirmus OF4.
abstract: OBJECTIVE
  To study crystallization and X-Ray diffraction of ectoine hydroxylase from Bacillus pseudofirmus OF4.
  
  
  METHODS
  We cloned the gene BpectD from B. pseudofirmus OF4 with a His6 tag and overexpressed it in E. coli BL21 (DE3). Then, we purified protein BpEctD by Ni2 -chelating affinity and size-exclusion chromatography. After that, crystals were grown by the sitting-drop vapour-diffusion method at 289 K and diffracted at 100K using an in-house X-ray source.
  
  
  RESULTS
  Protein BpEctD was expressed and purified successfully. We obtained well diffracting crystals of about 360 μm x 240 μm x 60 μm in size using a solution consisting of 0.2 mol/L magnesium chloride hexahydrate, 0.1 mol/L bis-tris pH6. 5, 25% (W/V) polyethylene glycol 3,350 at a protein concentration of 6. 5 mg/mL, and collected X-ray diffraction data to 2.40 Å resolution in the anorthic space group P1, with unit-cell parameters a = 45.18Å, b = 58.87Å, c = 68.81 Å, α = 77.48 degrees, β = 86.03 degrees, γ = 66.97 degrees. The asymmetric unit contains two molecules of BpEctD with a Mattews coefficient of about 2.44 Å3/Da and a solvent content of 49.53%.
  
  
  CONCLUSIONS
  According to the X-ray diffraction data, the three-dimensional structure of BpEctD from B. pseudofirmus OF4 soon will be analyzed, and it will provide insights into the biochemical properties of ectoine hydroxylase.
authors.forename: Jiangli
authors.initials: J
authors.lastname: Yang
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Wang
authors.forename: Shujing
authors.initials: S
authors.lastname: Xu
authors.forename: Wanyi
authors.initials: W
authors.lastname: Guan
authors.forename: Baohua
authors.initials: B
authors.lastname: Zhao
authors.forename: Hui
authors.initials: H
authors.lastname: Dong
authors.forename: Jiansong
authors.initials: J
authors.lastname: Ju
mesh_terms: D000595:Amino Acid Sequence; D000599:Amino Acids, Diamino / Q000378:metabolism*; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003460:Crystallization; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014961:X-Ray Diffraction
chemical_list: D000599:Amino Acids, Diamino; D001426:Bacterial Proteins; C045628:ectoine; D006899:Mixed Function Oxygenases