Computational protocol: A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase

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Protocol publication

[…] Crystals were screened using a Phoenix robot (Art Robbins Instruments) with the sitting-drop, vapor-diffusion method at 20 °C and drops containing 0.3 μL of protein solution plus 0.3 μL of reservoir solution. The following commercial screening kits were used: SaltRxTM, PEG/IonTM, IndexTM, Crystal ScreenTM, and PEGRxTM (all from Hampton Research). Crystals of both RmNag full-length and CTR appeared in a drop containing 0.1 M Bis-Tris (pH 5.5–6.5), 2 M (NH4)2SO4. Optimized crystals suitable for diffraction were grown in drops containing 1.5 μL of protein solution and 1.5 μL of reservoir solution (1.3–1.6 M (NH4)2SO4 , 0.1 M Bis-Tris pH 5.5–6.5) at 20 °C. Crystals were soaked in reservoir solution supplemented with 20% glycerol, and then vitrified in liquid nitrogen. Diffraction data of the CTR and full-length RmNag were collected at 100 K using beamline BL14.2 at BESSY (Berlin, Germany) and P11 at PETRA III (Hamburg, Germany), respectively. Indexing, integration and scaling of data were carried out with XDS. The program XPREP (Bruker) was used to further analyse and prepare datasets for structure solution and refinement. Statistics of the datasets are summarized in . [...] The crystal structure of the RmNag CTR was determined by the single-wavelength anomalous diffraction (SAD) method. Phase calculations and initial model building were carried out by the program AutoSol from the PHENIX suite. Thereafter, model building and refinement were performed using Coot and Refmac5, respectively. To determine the RmNag full-length structure, a model representing its NTR was prepared firstly by modifying the coordinates of Bacillus subtilis N-acetylglucosaminidase (PDB code: 2BMX), guided by an amino acid sequence alignment. The resultant NTR model, together with the model of CTR, were employed as search models in PHASER. The refinement was performed by Refmac5 with automatically determined TLS groups and NCS restraints (chain A to B) introduced. The structure was refined to 2.8 Å with Rwork and Rfree values of 0.229 and 0.254, respectively. The geometry of the final models was scrutinized using MolProbity. Structural homologs of CTR were identified at the DALI server. Secondary structure elements were identified employing DSSP. The structure cartoons were prepared in PyMOL (v.1.3; Schrödinger LLC). The sequence alignments were created with MUSCLE and ESPript. Coordinates and structure factors of RmNag have been deposited at the Protein Data Bank under accession number 4ZM6. […]

Pipeline specifications

Software tools XDS, PHENIX, Coot, REFMAC5, MolProbity, DALI, PyMOL
Applications Small-angle scattering, Protein structure analysis
Chemicals Glucosamine, Pantetheine