Computational protocol: Crystal structure of the P2 C-repressor: a binder of non-palindromic direct DNA repeats

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Protocol publication

[…] Crystals of selenomethionine substituted P2 C were obtained in sitting drops at 20°C by mixing 300 nl protein solution (6 mg/ml) with 100 nl precipitant using a Mosquito nanodrop crystallization robot. The precipitant solution contained 3.9 M sodium formate and 0.1 M Tris pH 7.5. Crystals shaped as long rods grew in a few days and were frozen in liquid nitrogen directly before data collection. Crystals of native P2 C were obtained from hanging drops at 20°C by mixing 2.4 μl protein solution with 0.8 μl precipitant solution containing 3.9 M sodium formate and 0.1 M Tris pH 7.5. Crystals grew as long rods in a few days and were thereafter flash frozen in liquid nitrogen. Diffraction data was collected at 100 K from flash-frozen crystals in liquid nitrogen at beamline BL14.1 at the Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung (BESSY). A SAD dataset was collected for the selenomethionine crystal at a wavelength of 0.980 Å. The native data set was collected at a wavelength of 0.978 Å. Both datasets were processed using MOSFLM () and SCALA in the CCP4 program suite (). Data collection and re?nement statistics are presented in . [...] The scaled selenomethionine data set was phased using Phenix Autosol (,), which also performed an automated initial model building. The structure given from Phenix Autosol was not further processed, but used to determine the phases for the native data set. Initial phases for the native data were obtained by molecular replacement in MOLREP () using the structure from the selenomethionine substituted P2 C. The model was automatically built using ARP/wARP () followed by cycles of manual building using coot () and refinement with refmac5 () in the CCP4 program suite () to complete the structure. TLS parameters were used during the last steps of refinement (). The electron density for the N-terminal methionine and the last 14 C-terminal residues could however not be observed. These residues were therefore omitted from the model. Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 2xcj. […]

Pipeline specifications

Software tools iMosflm, CCP4, PHENIX, Molrep, ARP/wARP, Coot, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli