Computational protocol: Crystal Structure of Talaromyces cellulolyticus (Formerly Known as Acremonium cellulolyticus) GH Family 11 Xylanase

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Protocol publication

[…] The selected crystals were placed in the cryoprotectant solution that consisted of 33 % (v/v) ethylene glycol, 0.67 M Bis-Tris (pH 5.5), 0.53 M sodium dihydrogen phosphate, and 0.53 M potassium dihydrogen phosphate. The soaked crystal was collected with a cryo-loop and flash-cooled under cryostream of nitrogen gas at −173 °C. X-ray diffraction data experiment was carried out at the SPring-8 BL44XU in Hyogo, Japan. The dataset was collected at a wavelength of 0.9 Å using a Rayonix MX225HE detector at the beamline. The distance from the crystal to the detector was 230 mm. The crystal was rotated at 200° with an oscillation angle of 0.5° per frame. The data collected from diffraction measurements were indexed, integrated, and scaled with the programs in the HKL-2000 software package []. [...] TcXylCΔN17 structure was solved by molecular replacement with MOLREP [] in the CCP4 package using as a search model the structure of XYNII from Trichoderma reesei (TrXyl) (Protein Data Bank (PDB; ID 1XYP) [] which shows 61 % sequence identity with TcXylCΔN17. The resulting electron density maps were used to refine and build a model of TcXylCΔN17. Structure model-building was performed with Coot []. The structure was refined using REFMAC5 []. Water molecules were introduced at peaks over 3.0 root-mean-square deviation (RMSD) in the difference Fourier map fulfilling reasonable interactions with the protein model. Ramachandran plot of the final structure was validated using ProCheck []. Molecular graphics images were generated using PyMOL ( Processing parameters are presented in Table . […]

Pipeline specifications

Software tools HKL-2000, Molrep, CCP4, Coot, REFMAC5, PROCHECK, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Thermogladius calderae