SUNY at Albany
June 19-23, 2001
Ribosome structure and function by single-particle cryo-EM
Electron microscopy of individual macromolecules ("single particles") is a very rapid technique for probing the three-dimensional ("3D") structure of biological macromolecules. The different orientations of the macromolecules in the vitreous-ice embedding medium are exploited for extracting 3D information from the microscopical data. The "angular reconstitution" approach has become a routine technique for finding 3D structures of macromolecules with arbitrary pointgroup symmetry, from the entirely asymmetric ribosomes to viruses with icosahedral symmetry.
Due to its practical advantages, the angular reconstitution cryo-EM approach is well suited for studying macromolecular systems in different conformational states. The highest resolution levels that have been achieved by the approach on ribosomal structures is ~7.5 Angstrom for the E. coli 50S ribosomal subunit (Matadeen et al., Structure 7 (1999) 1575-1583). Such resolution levels can thus already be obtained for ribosomes of all species and in any functional state one manages to lock the ribosome into. A new high-resolution structure is available of the full 70S E. coli ribosome, which allows the study of conformational changes occurring in the large subunit upon binding of the small subunit. It is anticipated that cryo-EM will eventually yield ribosomal structures at resolutions better than 4?. The problems we face in achieving this goal seem to be more associated with the logistics of the data processing than with the quality of the images obtained in the new generation of 300kV liquid-helium-cooled electron microscopes.
Rishi Matadeen, Tillmann Pape, Brent Gowen, Elena Orlova, Bruno Klaholz, Ardan Patwardhan, and Marin van Heel
Imperial College, Dept. of Biochemistry, London SW7 2AY, UK.