Cryo-electron microscopy and single-particle reconstruction , studying the mechanism of protein biosynthesis and other molecular processes in the cell.
Additional Website: http://www.hhmi.org/research/structure-ribosome-and-mechanism-protein-synthesis
We investigate the mechanism of translation on the ribosome by using cryo-electron microscopy and single-particle reconstruction aiming at the highest resolution. Using these methods, and flexible fitting of X-ray structures, the dynamics of the decoding and translocation mechanisms are revealed. The same methods can also be used to study a wide range of macromolecular complexes at close-to-atomic resolutions.
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 maps of EF-G(H94A)-bound E. coli 70S complexes. (A) Map of nonrotated ribosome (transparent) fitted with atomic models for both ribosome and EF-G. (B) Map of rotated ribosome at 3.6Å (transparent) fitted with atomic models. (C) Superimposition of the maps of the 30S subunit (green for rotated) when two maps are aligned on the 50S subunits. (D) Map-fitted structures of all tRNAs and EF-Gs at their respective positions shown in (C). (from Li et al. (2015) Science Advances).
- Sun, M., Li, W., Blomqvist, K., Das, S., Hashem, Y., Dvorin, J.D., and Frank, J. (2015). Dynamical features of the Plasmodium falciparum ribosome during translation. Nucleic Acids Res 43, 10515-10524.
- Des Georges, A., Dhote, V., Kuhn, L., Hellen, C.U., Pestova, T.V., Frank, J., and Hashem, Y. (2015). Structure of mammalian eIF3 in the context of the 43S preinitiation complex. Nature 525, 491–495.
- Chen, B., Kaledhonkar, S., Sun, M., Shen, B., Lu, Z., Barnard, D., Lu, T., Gonzalez, R.L., and Frank, J. (2015). Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryo-EM. Structure 23, 1097–1105.
- Li, W., Liu, Z., Koripella, R.K., Langlois, R., Sanyal, S., and Frank, J. (2015). Activation of GTP hydrolysis in mRNA-tRNA translocation by Elongation Factor G. Science Advances 1 e1500169.
- Zalk, R., Clarke, O.B., des Georges, A., Grassucci, R.A., Reiken, S., Mancia, F., Hendrickson, W.A., Frank, J., and Marks, A.R. (2015). Structure of a mammalian ryanodine receptor. Nature 517, 44–49.
- Dashti, A., Schwander, P., Langlois, R., Fung, R., Li, W., Hosseinizadeh, A., Liao, H.Y., Pallesen, J., Sharma, G., Stupina, V.A., Simon, A.E., Dinman, J., Frank, J., and Ourmazd, A. (2014). Trajectories of the ribosome as a Brownian nanomachine. Proc Natl Acad Sci U S A 111, 17492-17497.