Computational protocol: The Escherichia coli Cell Division Protein and Model Tat Substrate SufI (FtsP) Localizes to the Septal Ring and Has a Multicopper Oxidase Like Structure

Similar protocols

Protocol publication

[…] Crystals were grown by the vapour-diffusion method in sitting drops at 20 °C. Screens were set up with a crystallization robot (TECAN, UK). Crystallization drops were obtained by mixing 0.2 μl of protein solution in 20 mM Mops (pH 7.2), 200 mM NaCl with 0.2 μl of the crystallization screen and were equilibrated against 100 μl of mother liquor. SufI crystals were grown by mixing the protein solution at 0.75 mg/ml with EB Wizard I condition 37 (2.5 M NaCl, 100 mM imidazole, pH 8.0). Crystals were visible after 5 days with dimensions of approximately 50 μm × 100 μm × 200 μm.Diffraction data from the SufI crystals were collected at 100 K on beam lines ID23-1 and ID29 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The crystals were cryoprotected in 8 M sodium formate. The crystals belonged to either space group P21 or space group P212121, with both the monoclinic and orthorhombic crystals growing from the same condition, although the orthorhombic form required a longer growth period. All X-ray data integration and scaling were performed with the computer programs MOSFLM and Scala. The lattices of the two crystal forms were not found to be related. gives the crystallographic data collection and processing statistics. [...] Initial structure factor phases were obtained for the orthorhombic SufI crystals by molecular replacement. The program SCWRL 3.0 was used to model the SufI side chains onto the closely homologous blue copper oxidase CueO from E. coli (PDB ID 1PF3, 32% sequence identity) and the resulting model was used as a search model for molecular replacement with the computer program CCP4-PHASER. Two copies of the search model were found in the asymmetric unit and the SufI sequence was then built into the resulting density with X-fit, followed by multiple rounds of iterative refinement in BUSTER-TNT and model building in X-fit. Non-crystallographic symmetry (NCS) restraints were applied in BUSTER-TNT, with restraints lifted for individual residues where required. Once completed, the orthorhombic SufI structure was used as a search model for molecular replacement in the monoclinic crystal form, again with CCP4-PHASER. This provided a good solution with two copies in the asymmetric unit. The molecules were in a conformation very similar to those in the orthorhombic form with the exception of the loop around residues 55–66, which shifted between the two crystal forms. This region was removed from the search model and the molecular replacement search was repeated. Residues 55–66 were manually rebuilt into the resultant electron density with X-fit and then multiple rounds of iterative refinement with NCS in BUSTER-TNT, followed by model building in X-fit, were performed. The NCS for individual residues was lifted locally where required. Full details of the refinement parameters for both crystal forms can be found in . The anomalous difference density was calculated with the program CCP4-FFT and all superpositions were performed with the program CCP4-LSQKAB. […]

Pipeline specifications

Software tools CCP4, SCWRL
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Escherichia coli