Computational protocol: The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*

Similar protocols

Protocol publication

[…] Purified SeMet CtXyl5A-D was concentrated and stored in 5 mm DTT, 2 mm CaCl2. Crystals of seleno-l-methionine-containing protein were obtained by hanging drop vapor diffusion in 40% (v/v) 2-methyl-2,4-pentandiol. The data were collected on Beamlines ID14-1 and ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France) to a resolution of 2.64 Å. The data were processed using the programs iMOSFLM () and SCALA () from the CCP4 suite (Collaborative Computational Project, Number 4, 1994). The crystal belongs to the orthorhombic space group (P21212) (). The structure was solved by molecular replacement using independently solved structures of some of the modules of the CtXyl5A: CtGH5-CBM6 (PDB code 2y8k) (), Fn3 (PDB code 3mpc) (), and CtCBM62 (PDB codes 2y8m, 2yfz, and 2y9s) () using PHASER (). The CtCBM13 domain was built de novo. BUCCANEER () and PHENIX () were initially used for auto building. The structure was completed by iterative cycles of manual rebuilding in COOT () in tandem with refinement with RefMac5 (). The final values for Rwork and Rfree) were 23.73 and 27.80%) using TLS and restraining refinement to amino acid residues 36–373 representing the CtGH5 module, 374–516 for the CtCBM6, 517–652 for CtCBM13, and 653–742 for CtFn3. Stereochemistry was assessed with COOT () and PDBSUM () (with 677 residues (96%) in preferred, 22 in allowed regions (3%), and 6 outliers (1%) in the Ramachandran plot).To obtain structures of CtGH5-CBM6 in complex with ligand the protein was crystallized using the sitting drop vapor phase diffusion method with an equal volume (100 nl) of protein and reservoir solution (unless otherwise stated), using the robotic nanodrop dispensing systems (mosquitoR LCP; TTPLabTech). Crystals of the protein (10 mg/ml) co-crystallized with arabinose (300 mm) were obtained in 1 m ammonium sulfate, 0.1 m Bis-Tris, pH 5.5, and 1% PEG 3350. Crystals with xylose (300 mm) grew in 100 mm sodium/potassium phosphate, 100 mm MES, pH 6.5, and 2 m sodium chloride. To obtain crystals of the arabinoxylanase in complex with an oligosaccharide, the nucleophile mutant E279S was used and mixed with a range of arabinoxylooligosaccharides that was generated by digestion of WAX with CtGH5-CBM6 (see above) and thereafter by 100 nm of the Cellvibrio japonicus GH43 exo-1,4-β-xylosidase (). Only the inclusion of the largest purified oligosaccharide generated crystals of the arabinoxylanase. Crystals of CtGH5E279S-CBM6 were obtained by mixing an equal volume (100 nl) of the protein (11 mg/ml)/oligosaccharide (10 mm) solution and mother liquor solution consisting of 100 mm Tris-Bicine, pH 8.5, 12.5% (w/v) polyethylene glycol with an average molecular mass of 1,000 Da, 12.5% (w/v) polyethylene glycol with an average molecular mass of 3,350 Da and 12.5% (R,S)-2-methyl-2,4-pentanediol (racemic). Crystallographic data were collected on Beamlines IO2, IO4-1, and I24 at the DIAMOND Light Source (Harwell, UK). The data were processed using XDS () The crystal structures were solved by molecular replacement using MolRep () with CtGH5-CtCBM6 (PDB code 5AK1) as the search model. The refinement was done in RefMac5 (), and COOT () was used for model (re)building. The final model were validated using Molprobity (). The data collection and refinement statistics are listed in . […]

Pipeline specifications

Software tools iMosflm, CCP4, Buccaneer, Coot, REFMAC5, XDS, Molrep, MolProbity
Applications Small-angle scattering, Protein structure analysis
Chemicals Alanine, Arabinose, Xylose