New methods of image processing and three-dimensional (3-D) reconstruction have made it possible to obtain structural models of complex molecular assemblies such as ribosomes and hemocyanin molecules at 2-3 nm resolution from electron micrographs of non-crystalline specimens. This capability will be used in 3-D localizations of functionally important ligand-binding sites on the ribosome and mappings of specific proteins in ribosomes and hemocyanin. Many of the studies will be done in collaborations with other laboratories. We will also obtain precise models, to a resolution of 2-3 nm, of the subunit:subunit association in the 70S (prokaryotic) and 80S (eukaryotic) ribosomes by 3-D matching of reconstructed subunits and ribosomes. Specifically we will reconstruct: (1) the 50S subunit from E. coli depleted of the L7/L12 proteins which are involved in the elongation factor-dependent GTPase activities associated with translation, (2) a 50S-spuromycin-antibody complex in order to map the position of the peptidyl transferase center, (3) the 70S monosome from E. coli with and without bound tRNA, (4) a eukaryotic 40S subunit labeled with a monospecific antibody directed against one of the regulatory phosphorylation sites on protein S6, (6) a complex consisting of the 40S subunit and initiation factor eIF-3, and (7) a mammalian 80S ribosome. We will also reconstruct the 24-subunit hemocyanin molecule from Androctonus australis and one or more selected monoclonal immunocomplexes to localize specific subunits. These studies will be supported by methodological investigations and extended by the use of cryo-electron microscopy of frozen- hydrated, unstained specimens.
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