The translation elongation cycle-capturing multiple states by cryo-electron microscopy

Abstract

During the work cycle of elongation, the ribosome, a molecular machine of vast complexity, exists in a large number of states distinguished by constellation of its subunits, its subunit domains and binding partners. Single-particle cryogenic electron microscopy (cryo-EM), developed over the past 40 years, is uniquely suited to determine the structure of molecular machines in their native states. With the emergence, 10 years ago, of unsupervised clustering techniques in the analysis of single-particle data, it has been possible to determine multiple structures from a sample containing ribosomes equilibrating in different thermally accessible states. In addition, recent advances in detector technology have made it possible to reach near-atomic resolution for some of these states. With these capabilities, single-particle cryo-EM has been at the forefront of exploring ribosome dynamics during its functional cycle, along with single-molecule fluorescence resonance energy transfer and molecular dynamics computations, offering insights into molecular architecture uniquely honed by evolution to capitalize on thermal energy in the ambient environment.This article is part of the themed issue 'Perspectives on the ribosome'.

Keywords: free-energy landscape; mRNA–tRNA translocation; molecular machines; single-particle reconstruction.

Figure 1.

Ratchet-like motion and associated L1 stalk motion accompanying the movement of tRNA. Cryo-EM maps of Escherichia coli 70S·MFTI-tRNA^Ile–puromycin complexes before (a) and after (b) binding of EF-G•GDPNP (a non-hydrolysable GTP analogue) [26]. The L1 stalk of the 50S subunit is ‘open’ before (a) and closed after (b) binding of EF-G. (c) Conformational change of L1 stalk, which moves the tRNA from P/E (b) into E/E (a) position. Landmarks: L9, ribosomal protein L9; L2, protein L2; H76, helix 76 from the 23S rRNA; L1, L1 protuberance or stalk in the 50S subunit. (Reproduced with permission from [26].)

Figure 2.

Cryo-EM reconstructions of the P. falciparum 80S ribosomes purified from cell extract [93]. (a–d) Density maps of the P. falciparum 80S ribosome in non-rotated states. (a) Bound with E-tRNA (approx. 97 000 particles) at an average resolution of 4.7 Å; (b) bound with P-tRNA (approx. 14 500 particles) at 6.7 Å; (c) bound with P/P- and E/E-tRNAs (approx. 14 500 particles) at 6.7 Å and (d) without tRNAs (approx. 32 000 particles) at 5.1 Å. (e) Rotated state (approx. 23 000 particles) at 5.8 Å resolution; 60S subunits are coloured blue and 40S subunits are yellow. (f) Positions of tRNAs for all 80S complexes in (a–d). Structures and positions of E-, P- and P/E-tRNA were obtained by MDFF fitting, and the structure and position of A/P-tRNA was taken from the existing model, PDB 3J0Z, rigid-body fitted into the segmented map in UCSF Chimera. Contours of cryo-EM densities are displayed in mesh; structures of tRNA are displayed as ribbons and the mRNA path has been added as a cartoon. (Reproduced with permission from [93].)