Computational protocol: Structural and Molecular Basis for Resistance to Aminoglycoside Antibiotics by the Adenylyltransferase ANT(2″)-Ia

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Protocol publication

[…] ANT(2″)-Ia was concentrated to 60 mg/ml, and crystals were grown at 20°C from hanging drops using the vapor diffusion method. Optimized crystallization conditions for ANT2(2″)-Ia were 10% 2-propanol, 20% polyethylene glycol (PEG) 4000, and 0.1 M HEPES (pH 7.5). ANT(2″)-Ia was cocrystallized with kanamycin B (Sigma Aldrich, Oakville, ON, Canada) at a ratio of 5 times the molar concentration of the former. For data collection, the crystals were passaged through mother liquor containing 20% (vol/vol) glycerol and frozen in nitrogen.Data were collected at 100 K with Cu Kα X rays generated by a Rigaku 007 Microfocus rotating-anode generator equipped with VariMax HF optics and a Rigaku Raxis IV2+ detector. All X-ray data were reduced with HKL-3000 (). The ANT(2″)-Ia apoenzyme structure was solved by S-SAD using Phenix.solve (); four of the six sulfur sites (four cysteine residues) were located. Automated model building was performed with Phenix.autobuild, refinement was performed using Phenix.refine, and manual model building was performed with Coot (). The ANT(2″)-Ia–kanamycin B complex structure was solved by molecular replacement (MR) using the apoenzyme structure. Fo-Fc difference density corresponding to the aminoglycoside molecule was readily traceable after MR. Translation-libration-screw rotation (TLS) parameterization was utilized for refinement of both structures. Geometries were verified using the Phenix and Coot validation tools and the RCSB PDB deposition server. Occupancy values for solvent molecules and Mg2+ atoms were refined. All B factors were refined as isotropic. Average B factor and bond angle/bond length RMSD values were calculated using Phenix. [...] For comparison of similar structures, the Cα RMSD was calculated using the SSM superposition tool in Coot () and the Dali server (). The PDBePISA tool () was utilized to identify protein-ligand interactions. Structural homologs in the PDB were identified using the Dali server (). Secondary structure boundaries were generated using the PDBsum server (). […]

Pipeline specifications

Software tools SHELX, HKL-3000, PHENIX, Coot, DALI
Databases PDBsum
Application Protein structure analysis
Organisms Homo sapiens
Diseases Communicable Diseases, Infection
Chemicals Cyclic AMP, Adenosine Triphosphate, Aminoglycosides, Boron, Gentamicins, Kanamycin, Magnesium, Nucleosides, Tobramycin