Computational protocol: Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6*

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Protocol publication

[…] The crystals of LigD, LigO, LigL, and LigG were placed in a reservoir solution containing 10–20% (v/v) glycerol and then flash-cooled in liquid nitrogen. The x-ray data sets for LigD, LigO, LigL, and LigG were collected at the Berkeley Center for Structural Biology beamlines 8.2.1 and 8.2.2 of the Advanced Light Source at Lawrence Berkeley National Laboratory. Data sets were indexed and scaled using HKL2000 (). LigD, LigO, and LigL crystal structures were determined by the molecular replacement method with the program PHASER () within the Phenix suite (). LigD was solved using as a search model the Protein Data Bank coordinates of 3IOY, which has 35% sequence identity with the target. The LigO and LigL crystal structures were solved using the LigD coordinates as the search model. The results from the molecular replacement for LigD, LigO, and LigL showed a translation function Z-score of 15.1, 20.7, and 21.2, respectively, where translation function Z-score values of >8 strongly suggest correct solutions (). The atomic positions obtained from molecular replacement and the resulting electron density maps were used to build the LigD, LigO, and LigL structures and initiate crystallographic refinement and model rebuilding. The crystal structure of LigG was solved using selenomethionine-labeled protein by single-wavelength anomalous dispersion methods () with the phenix.autosol () and phenix.autobuild () programs. The figure of merit from single-wavelength anomalous dispersion phasing was 0.54, indicating a good phase quality (). Structural refinement was performed using the phenix.refine program (). Manual rebuilding using COOT () and the addition of water molecules allowed construction of the final models. Root mean square deviation differences from ideal geometries for bond lengths, angles, and dihedrals were calculated with Phenix (). The overall stereochemical quality of all final models was assessed using the program MOLPROBITY (). Superposition of the models for structural comparison was performed using COOT (). […]

Pipeline specifications

Software tools PHENIX, Coot, MolProbity
Application Protein structure analysis
Organisms Sphingobium sp. SYK-6
Chemicals Carbohydrates, Glutathione, NAD