Wednesday, May 4, 2011

Ribosome-SecYEG visualized in a membrane environment

A sub-nanometer cryo-EM reconstruction of the ribosome-SecYEG complex has been reported in the latest issue of Nature Structural & Molecular Biology. MDFF was employed to interpret the cryo-EM data at atomic level. The work is a collaboration between the Schulten (University of Illinois at Urbana-Champaign, USA) and Beckmann (University of Munich, Germany) groups.

Cryo-EM structure of the ribosome-SecYE complex in the membrane environment.
Jens Frauenfeld, James Gumbart, Eli O. van der Sluis, Soledad Funes, Marco Gartmann, Birgitta Beatrix, Thorsten Mielke, Otto Berninghausen, Thomas Becker, Klaus Schulten, and Roland Beckmann. Nat. Struct. Mol. Biol., 18, 614-621, 2011.

The ubiquitous SecY–Sec61 complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent-solubilized Sec complexes have been reported. Here we present a single-particle cryo-EM structure of the SecYEG complex in a membrane environment, bound to a translating ribosome, at subnanometer resolution. Using the SecYEG complex reconstituted in a so-called Nanodisc, we could trace the nascent polypeptide chain from the peptidyltransferase center into the membrane. The reconstruction allowed for the identification of ribosome–lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the protein-conducting channel (PCC). On the basis of our map and molecular dynamics simulations, we present a model of a signal anchor–gated PCC in the membrane.

Insights into translational fidelity

In a recent work published by the EMBO Journal, images of the ribosome bound to either a cognate or a near-cognate tRNA were obtained by cryo-electron microscopy, and MDFF was employed to generate atomic models, shedding light into the mechanisms by which the ribosome discriminates between correct and incorrect tRNAs. The work is a collaboration between the Schulten (University of Illinois at Urbana-Champaign, USA) and the Frank (Columbia University, USA) groups.

Structural insights into cognate vs. near-cognate discrimination during decoding.
Xabier Agirrezabala, Eduard Scheiner, Leonardo G. Trabuco, Jianlin Lei, Rodrigo F. Ortiz-Meoz, Klaus Schulten, Rachel Green, and Joachim Frank. EMBO J., 30, 1497-1507, 2011.

The structural basis of the tRNA selection process is investigated by cryo-electron microscopy of ribosomes programmed with (UGA/stop) codons and incubated with ternary complex containing the near-cognate Trp-tRNA{Trp} in the presence of kirromycin. Going through more than 350,000 images and employing image classification procedures, we find 8% in which the ternary complex is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near-cognate aa-tRNA with respect to EF-Tu and the ribosome, as well as the domain movements of the ribosome. The data bring direct structural insights into the induced fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa-tRNA and EF-Tu and comparison with the cognate case (UGG codon) offers clues as to how the conformational signals conveyed to the GTPase differ in the two cases.