Computational protocol: Lipoxygenase 2 from Cyanothece sp. controls dioxygen insertion by steric shielding and substrate fixation

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Protocol publication

[…] Diffraction data to 1.8 Å resolution were recorded from a single crystal on beamline BL-14.1 at BESSY II (Berlin, Germany) equipped with MAR CCD detector (MOSAIC 225) and processed and scaled with the XDS package. The oscillation images could be processed in either space group P222 or P422 with almost identical results (Rmerge = 7.1% in P222, 7.1% in P422) due to very similar lengths of two unit cell parameters. The hypothesis that the crystal was twinned was tested by a statistical analysis using phenix. xtriage. The results of the L-test indicated that no twinning was suspected. The Stanley factor 〈I2〉/〈I〉, , was found to be 1.865 (standard values of the 〈I2〉/〈I〉2 ratio are ∼2.0 for untwinned data and ∼1.5 for twinned data). Statistics for the data in P222 are presented in Table . [...] The crystal structure of holo CspLOX2 was solved by Molecular Replacement method (MR) with PHASER program using 7 highest scored templates identified by HHPRED server as the search models (PDB ids: 1f8n, 1lox, 2iuj, 2iuk, 3dy5, 3fg1, 3o8y). Initial attempts to solve the structure by MR were carried out in the tetragonal space group P43212 with one molecule occupying the asymmetric unit. The best fitting model was selected based on crystallographic R factors upon refinement of seven individual MR solutions in PHENIX. The model with lowest R factors derived from homo sapiens 5-lipoxygenase (PDB id: 3o8y) was remodeled by combining homology modeling with density-guided energy optimization as implemented in the Rosetta package and extensively refined in PHENIX and remodeled manually in Coot. The best model in P43212 space group could be refined to R = 36.2% and Rfree = 41.1%.The failure to refine the structure successfully in a tetragonal system indicated over-merging of diffraction data and implicated orthorhombic symmetry of the crystal. Molecular Replacement was carried in PHASER against orthorhombic diffraction data using the search model derived from homo sapiens 5-lipoxygenase (PDB id: 3o8y) and resulted in placing two CspLOX2 molecules in the asymmetric unit. Those two molecules are related by a rotation of 179.8 degrees around an axis mimicking a diagonal 2-fold axis in the tetragonal system, what explained the observed pseudo tetragonal symmetry. The initial model was remodeled with Rosetta using the calculated electron density map as restraints and further manually rebuild and refined in Coot and PHENIX, respectively. The final model consisting of residues 1 to 569, two Fe(II) ions, 1069 water molecules, 3 1-butanol molecules, 6 1,2-propanediol molecules and 1 glycerol molecule was refined at 1.8 Å resolution to crystallographic R and Rfree factors of 16.14% and 19.50%, respectively. The two CspLOX2 molecules exhibit high structural similarity as judged based on calculated root mean square deviation (RMSD) of 0.25 Å calculated for 565 compared Cα atoms. The stereo chemical quality of the structure was validated using PHENIX implementation of MolProbity. The crystal structures of L304V and L304F variants and Mn-CspLOX2 were solved using the Difference Fourier Method using the protein part of holo CspLOX2 structure as the starting model. All structures were refined and manually rebuild in PHENIX and Coot, respectively. Data collection, structure refinement statistics and validation are summarized in Table . […]

Pipeline specifications

Software tools XDS, PHENIX, HHPred, Coot, MolProbity
Applications Small-angle scattering, Protein structure analysis
Chemicals Carbon, Fatty Acids, Oxygen, Linoleic Acid