Computational protocol: Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state

Similar protocols

Protocol publication

[…] Ribosome complexes with mRNA and tRNAs were formed by mixing 5 μM Thermus thermophilus 70S ribosomes with 10 μM mRNA and incubation at 55°C for 10 min, followed by addition of 20 μM P-site (fMet-tRNAifMet) and 20 μM A-site (Phe-tRNAPhe) substrates (,). Each of the last two steps was allowed to reach equilibrium for 10 min at 37°C. For co-crystallization of MADU with the ribosome, the antibiotic was added to a final concentration of 200 μM, and the complex was left at room temperature for an additional 15 min prior to crystallization. All Tth 70S ribosome complexes were formed in the buffer containing 5 mM HEPES–KOH (pH 7.6), 50 mM KCl, 10 mM NH4Cl, and 10 mM Mg(CH3COO)2, and then crystallized in the buffer containing 100 mM Tris–HCl (pH 7.6), 2.9% (w/v) PEG-20K, 7–12% (v/v) MPD, 100–200 mM arginine, 0.5 mM β-mercaptoethanol (). Crystals were grown by the vapor diffusion method in sitting drops at 19°C and stabilized as described previously (,,) with 100 μM MADU included in the cryo-protection buffers. Diffraction data were collected using beamline 24ID-C at the Advanced Photon Source. All crystals belonged to the primitive orthorhombic space group P212121 with approximate unit cell dimensions of 210 Å × 450 Å × 620 Å and contained two copies of the 70S ribosome per asymmetric unit. Each structure was solved by molecular replacement using PHASER from the CCP4 program suite (). The search model was generated from the previously published structure of T. thermophilus 70S ribosome with bound mRNA and tRNAs (PDB entry 4Y4P from ()). The initial molecular replacement solutions were refined by rigid body refinement with the ribosome split into multiple domains, followed by positional and individual B-factor refinement. The final models of the 70S ribosome in complex with MADU and mRNA/tRNAs was generated by multiple rounds of model building in COOT (), followed by refinement in PHENIX (). The statistics of data collection and refinement are compiled in . […]

Pipeline specifications

Software tools CCP4, Coot, PHENIX
Application Protein structure analysis
Chemicals Alanine, Streptogramin A