Journal Article

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

Ficner, Ralf, Neumann, Piotr, Konevega, Andrey L., Rodnina, Marina V., Stark, Holger, Bock, Lars V., Fischer, Niels

2015

Overview

A single particle cryo-EM structure of the 70S ribosome in complex with the elongation factor Tu breaks the 3 Å resolution barrier of the technique and locally exceeds the resolution of previous crystallographic studies, revealing all modifications in rRNA and explaining their roles in ribosome function and antibiotic binding. Ribosome–EF-Tu complex structure One of the cell's largest and most important macromolecular complexes, the ribosome has been the target of intensive structural study. Until now, crystallographic studies have provided the highest resolution images of this complex. Now Holger Stark and colleagues have used the latest single-particle electron cryomicroscopy approaches to characterize the Escherichia coli 70S ribosome bound to the Tu elongation factor, a charged tRNA, and the antibiotic kirromycin, at a resolution that locally exceeds that obtained crystallographically. Novel insights are obtained about the modifications occurring on the rRNA and about the more flexible regions of the protein that are inaccessible to crystallographic analysis. 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 1 , 2 and all structures available to date have been limited to resolutions above 3 Å 3 , 4 . 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 2.65–2.9 Å resolution using spherical aberration (C s )-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome 5 (2.8 Å), but provides more detailed information (2.65 Å) 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 101/28 / 631/326/41/2536 / 631/337/574/1789 / 631/45/535/1258/1259 / 631/535/1258/1259 / Anti-Bacterial Agents - chemistry / Anti-Bacterial Agents - metabolism / Antibiotics / Cryoelectron Microscopy - methods / Crystal structure / Crystallography / E coli / Escherichia coli - chemistry / Escherichia coli - ultrastructure / Humanities and Social Sciences / Instrumentation / letter / Ligands / Models, Molecular / multidisciplinary / Peptide Elongation Factor Tu - chemistry / Peptide Elongation Factor Tu - metabolism / Peptide Elongation Factor Tu - ultrastructure / Pyridones - chemistry / Pyridones - metabolism / Ribonucleic acid / Ribosomes - chemistry / Ribosomes - metabolism / Ribosomes - ultrastructure / RNA / RNA, Bacterial - chemistry / RNA, Bacterial - metabolism / RNA, Bacterial - ultrastructure / RNA, Ribosomal - chemistry / RNA, Ribosomal - metabolism / RNA, Ribosomal - ultrastructure / RNA, Transfer - chemistry / RNA, Transfer - metabolism / RNA, Transfer - ultrastructure / Science