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EF-G-ribosome pre-translocation complex Frealign parameter files

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EF-G-ribosome pre-translocation complex Frealign parameter files

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Structure of the ribosome with elongation factor G trapped in the pretranslocation state


During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by a universally conserved ribosome-dependent GTPase [elongation factor G (EF-G) in prokaryotes and elongation factor 2 (EF-2) in eukaryotes]. Although the high-resolution structure of EF-G bound to the posttranslocation ribosome has been determined, the pretranslocation conformation of the ribosome bound with EF-G and A-site tRNA has evaded visualization owing to the transient nature of this state. Here we use electron cryomicroscopy to determine the structure of the 70S ribosome with EF-G, which is trapped in the pretranslocation state using antibiotic viomycin. Comparison with the posttranslocation ribosome shows that the small subunit of the pretranslocation ribosome is rotated by ∼12° relative to the large subunit. Domain IV of EF-G is positioned in the cleft between the body and head of the small subunit outwardly of the A site and contacts the A-site tRNA. Our findings suggest a model in which domain IV of EF-G promotes the translocation of tRNA from the A to the P site as the small ribosome subunit spontaneously rotates back from the hybrid, rotated state into the nonrotated posttranslocation state.

Data description

The archive linked to the right contains Frealign parameter files and scripts that were used to calculate 15 classes from 1.3 million single particle images of an E. coli EF-F-ribosome preparation. The data was collected at 300 kV on an FEI Titan Krios microscope and Falcon I direct electron detector at a calibrated magnification of 133,333, giving a pixel size on the specimen of 1.04 Å. The particle stack has 512 GB (in MRC/CCP4 format) and is available upon request as it is too large to be hosted on this web page.

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