Computational protocol: PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal

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Protocol publication

[…] Protein samples of wild-type PqsBC and the active site protein variant PqsBCC129A in 40 mm MOPS buffer, pH 8.1, 100 mm NaCl, 0.3 mm DTT at protein concentrations of 31.5 and 36.5 mg ml−1, respectively, were diluted with 100 mm NaCl to give 3 mg/ml. These samples were subjected to screens, including Pi-PEG, Procomplex, classics lite, Mbclass, Index, PACT, Morpheus, JCSG+, and MIDAS, using the sitting drop vapor diffusion method (Mosquito robot, TTP Labtech, Melbourne, UK). Crystals of PqsBCC129A resulted from the conditions of 0.2 m NaCl, 0.1 m Tris, pH 8.5, 25% PEG 3350 within 24 h, whereas octanoylated or apo-forms of wild-type PqsBC did not form crystals. Optimization was performed using 24-well sitting drop plates varying PEG in 1% increments, 23–27%, as well as Tris, pH 8.3–8.7. The final optimized crystallization condition was 25% PEG 3350, 0.1 m Tris, pH 8.3, and 0.2 m NaCl with 3 mg ml−1 protein concentration and a total volume of 4 μl. PqsBCC129A crystals were cryoprotected in a solution of 27% PEG 3350, 0.1 m Tris, pH 8.3, 0.2 m NaCl, 25% glycerol, and flash-cooled in liquid nitrogen prior to data collection at the Diamond Light Source synchrotron facility beamline I02 to 2.0 Å resolution with space group P212121 and a unit cell of a = 78.5 Å, b = 115.0 Å, and c = 289.7 Å, α = 90°, β = 90°, and γ = 90° (). This unit cell was predicted to have four PqsB and four PqsC polypeptides in the asymmetric unit with ∼50% solvent content. Molecular replacement was performed with PHASER using a variety of templates generated using MrBUMP () from PDB codes 3h76, 3h77, and 3h78 of the PqsD structure (). Using different resolution cutoffs, this resulted in an incomplete solution from which a partial model was built using BUCCANEER (), and electron density maps were subsequently improved using 4-fold non-crystallographic symmetry and solvent flattening with the CCP4 programs DM () and PARROT (). The resulting density-modified 2 Å electron density map was of high quality allowing 95% of the model to be built with BUCCANEER. The model was completed manually using COOT and refined with REFMAC5 () giving 2,474 residues in total, and crystallographic statistics are listed in (PDB code 5DWZ). Values for surface area buried were calculated using PISA (). […]

Pipeline specifications

Software tools Buccaneer, CCP4, REFMAC5
Application Protein structure analysis
Organisms Pseudomonas aeruginosa