Computational protocol: Klebsazolicin inhibits 70S ribosome by obstruction of the peptide exit tunnel

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Protocol publication

[…] High-resolution mass spectra were recorded on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (Varian 902-MS) equipped with the 9.4 T magnet (FTMS) in positive MALDI mode. The instrument was calibrated using the ProteoMass Peptide MALDI-MS Calibration Kit (Sigma-Aldrich). The accuracy of the mass peak measurements was 2.5 ppm. Samples (0.5 μl) were spotted on a steel plate with 0.5 μl of a 2,5-Dihydroxybenzoic acid matrix (Sigma-Aldrich) and air-dried at room temperature.Fragment ion spectra were recorded on an AB SCIEX TOF/TOF 5800 MALDI mass-spectrometer. The instrument was calibrated using the Mass Standards Kit for Calibration of AB Sciex TOF/TOF Instruments (AB Sciex). Samples (0.5 μl) were spotted on a steel plate with 0.5 μl of α-cyano-4-hydroxycinnamic acid matrix solution (AB Sciex) and air-dried at room temperature. Fragment ion spectra were generated by collision-induced dissociation (CID) in MS/MS mode. The accuracy of the mass peak measurements was 30 ppm for parent ions and 0.2 Da for daughter ions. Analysis of the MS and MS/MS data was carried out manually by GPMAW4.04 software (Lighthouse Data). [...] Ribosome complexes with mRNA and tRNAs were formed by programming of 5 μM Tth 70S ribosomes with 10 μM mRNA and incubation at 55°C for 10 min, followed by addition of 20 μM P-site (fMet-tRNAiMet) 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. The 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 the KLB included in the stabilization buffers (200 μM KLB). 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 210Å x 450Å x 620Å 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 KLB 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 GPMAW, CCP4, Coot, PHENIX
Applications MS-based untargeted proteomics, Protein structure analysis
Chemicals Macrolides