Computational protocol: Neisseria conserved hypothetical protein DMP12 is a DNA mimic that binds to histone-like HU protein

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Protocol publication

[…] For crystallization, 2 µl of the DMP12 solution (20 mg/ml; 20 mM Tris, pH 8.0, and 100 mM NaCl) was mixed with 2 µl of a reservoir containing 0.2 M magnesium acetate, 0.1 M sodium cacodylate, pH 6.5, and 20% (w/v) PEG 8000 (polyethylene glycol 8000) as a precipitant. Equilibration with the reservoir was achieved by the sitting drop method. Before flash-cooling, ethylene glycol at a final concentration of 15% was added as a cryoprotectant. After soaking the DMP12 crystals with 1 mM AuCl3 (Sigma-Aldrich) for 30 min, both native and SAD (single-wavelength anomalous dispersion) X-ray diffraction data were collected on beamline 13B at the National Synchrotron Radiation Research Center in Hsinchu, Taiwan. The data were processed using HKL2000 (). The space group of the native and AuCl3 soaked-DMP12 crystals was tetragonal I4. Unit cell dimensions for the native DMP12 crystal and AuCl3 soaked-DMP12 are given in . The DMP12 structure was determined using the SAD phasing method and the programs Shelx CDE and Phaser (,). High remote data in the range of 30–2.9 Å resolution collected at the wavelengths of 1.02243 Å were used. Four Au sites were located in an asymmetric unit (His 4 and His 117 from two monomers). The initial model was produced by the program BUCCANEER () and COOT () and revealed that each asymmetric unit contained two DMP12 molecules. The phase was further extended to 1.85 Å by using the native data set, and the programs COOT () and Refmac () were used for the subsequent refinement. Statistics for the data collection and refinement are shown in . The CCP4 package () and Pymol program () were used for the structural analyses and also for figure production. […]

Pipeline specifications

Software tools SHELX, Buccaneer, Coot, CCP4, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli