Abstract
Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8Å), but provides more detailed information (2.65Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.
Highlights
OH f13D Electron Cryomicroscopy Group, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany. 2Abteilung Molekulare Strukturbiologie, Institut fur Mikrobiologie und Genetik, GZMB, Georg-August Universitat Gottingen, Justus-von Liebig Weg 11, 37077 Gottingen, Germany. 3Molecular and Radiation Biophysics Department, B.P
Single particle electron cryomicroscopy has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis
The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes
Summary
13D Electron Cryomicroscopy Group, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany. 2Abteilung Molekulare Strukturbiologie, Institut fur Mikrobiologie und Genetik, GZMB, Georg-August Universitat Gottingen, Justus-von Liebig Weg 11, 37077 Gottingen, Germany. 3Molecular and Radiation Biophysics Department, B.P. The final 3D map has a similar, or locally even better resolution compared to the available X-ray structures of the E. coli ribosome (Fig. 1 and Extended Data Fig. 4a). And peptidyl-transferase activity of the 50S subunit require a network of six modified nucleosides at the entry site of the peptide exit tunnel (Fig. 2c)[17] Some of these modifications modulate antibiotic sensitivity of the ribosome, which can be explained at a structural level. Whereas the overall architecture of the EF-Tu–GDP–kirromycin–Phe–tRNAPhe complex is similar to that reported by X-ray crystallography[6,26], there are local differences in the conformation of the distorted tRNA in the A/T state, in the orientation of the catalytic His[84] residue and in the interactions between domain 2 of EF-Tu and the 30S subunit (Extended Data Fig. 7).
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