Computational protocol: Structural Characterization of a Novel Chlamydia pneumoniae Type III Secretion-Associated Protein, Cpn0803

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Protocol publication

[…] All Cpn0803 crystals were grown at 18°C using the hanging-drop vapour diffusion method. Equal volumes of Cpn0803 (3.0 mg/mL) in storage buffer (0.12 M potassium chloride, 0.2 M Tris pH 7.0) were mixed with crystallization solution (0.2 M potassium nitrate, 20% w/v PEG 3350) and dehydrated against 500 uL of 1.5 M ammonium sulfate. Cpn0803-SeMet crystals were grown in the same manner by streak seeding with native crystals and gradually increasing the concentration of ammonium sulfate in the well solution to 2.0 M.Native and SAD (SeMet) data sets were collected at 1.1 and 0.979 Å wavelengths on beamlines ×29 and ×25 respectively of the National Synchrotron Light Source at Brookhaven National Laboratory. Data sets were processed and scaled with HKL2000 to 2.0 and 2.3 Å, respectively . Of the 12 SeMet sites expected based on primary sequence, 11 were located by AutoSol in the anomalous data set. The Phenix software package was used to calculate initial phase information to generate an experimental map . A preliminary model was built which was then used for Molecular Replacement into the higher resolution, native data set of Cpn0803 using AutoMR. Model building and structure refinement of Cpn0803 was carried out through multiple iterations of Phenix-Refine and manual manipulation in COOT until the values of R and Rfree converged and geometry statistics reached acceptable ranges , . Surface area calculations were performed with the PDBe PISA server and volume calculations were performed using Chimera and the CASTp server , . All structural illustrations were generated with PyMol . Crystal structure similarity predictions were obtained using the DALI server . […]

Pipeline specifications

Software tools PHENIX, Coot, CASTp, PyMOL, DALI
Databases PDBe
Application Protein structure analysis
Organisms Chlamydia pneumoniae, Escherichia coli, Chlamydia trachomatis
Diseases Chlamydia Infections
Chemicals Phosphatidic Acids, Phosphatidylinositols