Computational protocol: On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase

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Protocol publication

[…] CRTI was concentrated to 9.5 mg ml−1 and crystallization conditions were screened using commercially available kits (Hampton Research and Qiagen suites). Experiments were carried out using the sitting-drop vapor-diffusion method in 96-well plates at 290 K. 200 nl protein solution and 200 nl precipitant solution were equilibrated against 50 µl reservoir solution. After optimization, diffraction quality crystals (200×150×150 µm) were obtained from hanging-drop vapor diffusion experiments in 24-well VDX plates (Hampton Research), where 2 µl of protein was mixed with 2 µl reservoir solution and equilibrated against 0.5 ml of reservoir.Crystals were obtained with 8% PEG 8K, 0.1 M NaCl, 0.1 M Na/K phosphate pH 6.2 and crystals of the selenomethionine substituted protein were obtained with 6% PEG 8K, 0.1 M NaCl, 0.1 M Na/K phosphate pH 6.2. Crystals were transferred to cryoprotection buffer (6–8% PEG 8K, 0.1 M NaCl, 0.1 M Na/K phosphate pH 6.2, 20% ethylene glycol) and flash frozen in liquid nitrogen. Data were collected at 100K at the Proxima 1 beamline of Synchrotron Soleil, France, using a Quantum 315r CCD detector (ADSC, USA). Few crystals yield diffraction data better than 2.5 Å resolution using synchrotron radiation. Native data were collected to 2.35 Å and data were collected to 3.0 Å from selenomethionine substituted crystal at the absorption peak (0.9791 Å), inflection point (0.9794 Å), and a remote wavelength (0.9770 Å). All data were indexed, integrated and scaled using HKL2000 . Data collection and refinement statistics are summarized in . Initial phase determination was performed by the MAD method using the program SHARP followed by solvent flattening in SOLOMON . Examining the electron density maps allowed the choice of space group as P3221. The program BUCCANEER was used to automatically locate structural fragments and a large part of the remaining model could be constructed in COOT . 1299 reflections (4.9%) were randomly selected for Rfree calculation. Refinement was carried out in REFMAC5 initially against the Se-Met peak data, with the resolution being increased in stages to 2.35 Å for refinement against the native data. The final stages of refinement were carried out in BUSTER and PHENIX . The structure was refined using three TLS domains. After each round of refinement the structure was validated with MOLPROBITY and the final structure validated with PROCHECK . All other crystallographic calculations were carried out with the CCP4 suite . [...] FAD, isoalloxazine and carotene substrates were docked into the crystal structure using the AUTODOCK 4.0 software . The docking parameters were calculated using the AUTODOCK standard procedures. Potential grid maps were calculated with a cubic box and a distance between grid points of 0.375 Å in targetdock and 0.5 Å in blinddock calculations. “Blinddock” simulations refer to calculations in which the grid box encompassed the entire protein. In “targetdock” simulations the grid box was narrowed down stepwise to a protein domain previously identified in blinddock experiments. The Lamarckian Genetic Algorithm with 25 million energy evaluations was applied as a search method. 200 docking experiments were carried out in each simulation. The docking results were evaluated by cluster analysis at a root mean square deviation cutoff of 2 Å. Structures were visualized using the PyMol Molecular Grapics System, version 1.3, Schrödinger, LLC. […]

Pipeline specifications

Software tools AutoDock, PyMOL
Application Protein interaction analysis
Organisms Pantoea ananatis, Oryza sativa
Diseases Malnutrition
Chemicals Oxygen, Phosphatidylcholines, Quinones, Vitamin A