Computational protocol: Structural Insight into the Specific DNA Template Binding to DnaG primase in Bacteria

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Protocol publication

[…] Initial crystallization was carried out by hanging drop vapor diffusion at 18 °C, using the crystallization screen kits from Hampton Research. The purified BsuDnaG was concentrated to 10 mg/ml in 25 mM Tris pH 8.0, 100 mM NaCl and 1 mM DTT for crystallization trials. A total of 1 μl protein solution was mixed with 1 μl well solution and equilibrated against 200 μl reservoir solution. Crystals were observed after two months in reservoir solution of 0.2 M sodium citrate tribasic dehydrate, 0.1 M Tris hydrochloride (pH 8.5) and 30% (w/v) polyethylene glycol 400. Before data collection, the crystals were cryoprotected by the addition of 20% (v/v) glycerol and flash frozen in liquid N2.X-ray diffraction data of BsuDnaG crystals were collected at 100 K using beam line BL17U at Shanghai Synchrotron Radiation Facilities (SSRF). Data sets were processed and scaled by HKL2000. The phase and the initial model of BsuDnaG were obtained by molecular replacement method using a polyalanine model of DnaG RPD domain (PDB 4e2k) from Staphylococcus aureus. The residues 116–363 and residues 367–428 were searched separately by using Phenix.AutoMR. Coot and Phenix.refine were used for manually building and refinement, respectively. The qualities of the final models were checked with the program MolProbity. Details of the overall refinement and final quality of the models were shown in Table . The program PyMOL (http://www.pymol.sourceforge.net/) was used to prepare structural figures. […]

Pipeline specifications

Software tools PHENIX, Coot, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Bacteria, Escherichia coli, Bacillus subtilis, Bacillus subtilis BEST7003, Geobacillus stearothermophilus
Chemicals Zinc