We investigate the mechanism of translation on the ribosome by using cryo-electron microscopy and single-particle reconstruction.Using these methods, and flexible fitting of X-ray structures, the dynamics of the decoding and translocation mechanisms are revealed.

Our laboratory conducts research on the mechanism of translation by the ribosome and on other processes involving molecular machines in the cell.The primary method of structural research is cryo-electron microscopy, based on the principle of forming a three-dimensional image by collecting and combining thousands of projections of the molecules embedded in a thin layer of ice.This method of “single-particle reconstruction” was pioneered in our lab, and is now widely used to study macromolecular interactions in a large range of systems.

To this end, well-characterized, functionally active complexes are prepared in vitro.They are stalled by chemical means (antibiotics, GTP nonhydrolyzable analogs, etc.), placed on a grid, and rapidly frozen by immersion into liquid ethane at liquid-nitrogen temperature.Images are recorded either on film (to be subsequently scanned) or electronically by means of a CCD camera.The resulting images are subsequently processed in the computer using the software system SPIDER and other, ancillary software, resulting in a three-dimensional density map.As the resolution of this map falls short of the atomic scale (i.e., 3Å and below), it is necessary to interpret the maps by flexible fitting of components whose structure has been solved by X-ray crystallography or NMR.

In the application to the study of the ribosome and its interactions with mRNA, tRNA and a variety of factors during translations, we have made important discoveries about the dynamics of initiation, decoding, translocation, termination, and recycling in both prokaryotic and eukaryotic systems.The highest resolution of a functional bacterial ribosome complex that has been achieved is 6.7Å (see Fig.1).We continue to make efforts to advance the resolution by improving specimen preparation, data collection, and classification.

Fig. 1.Cryo-EM map of a complex showing the aminoacyl-tRNA (purple) in the process of entering the E. coli ribosome, still engaged with EF-Tu (red), and stalled by the interference of kirromycin, an antibiotic (not seen).The large subunit is painted blue, the small subunit yellow.tRNAs in the P-site (green) and E-site (orange) positions are also seen.A new method of flexible fitting, Molecular Dynamics-based Flexible Fitting (MDFF), was used to produce the atomic model embedded in the semi-transparent density.MDFF was developed by our collaborator Klaus Schulten (University of Illinois in Urbana-Champaign), employing molecular dynamics simulations (Trabuco et al. K., Structure *16*, 673-683).