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. We continue to make efforts to advance the resolution by improving specimen preparation, data collection, and classification.
Derek J. Taylor, Jakob Nilsson, A. Rod Merrill , Gregers Rom Andersen , Poul Nissen, and Joachim Frank (2007) Structures of modified eEF2 . 80S ribosome complexes reveal the role of GTP hydrolysis in translocation. EMBO J. 26:2421–2431.
Haixiao Gao, Andrey Zavialov, Richard Gursky, Suparna Sanyal, Mans Ehrenberg, Joachim Frank, and Heiwei Song (2007) RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors. Cell 129:929-941.
Joachim Frank, Haixiao Gao, Jayati Sengupta, Ning Gao, and Derek J. Taylor (2007) The process of mRNA-tRNA translocation. PNAS 104:19671-19678.
Sjors H.W. Scheres, Haixiao Gao, Mikel Valle, Gabor T Herman, Paul P.B. Eggermont, Joachim Frank , and Jose-Maria Carazo Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nature Methods 4:27-29.
Leonardo G. Trabuco, Elizabeth Villa, Kakoli Mitra, Joachim Frank, and Klaus Schulten (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure, in press.