The long term goal of this project is to provide a structural model for the protein biosynthetic machinery of the cell. The objective of this application is to determine the structure of the large ribosomal subunits from B. stearothermophilus at medium resolution. Since the ribosomes are of enormous size, complex asymmetric structure, somewhat flexible and instable, the determination of their structure using diffraction methods is expected to require application of extensive resources and a high level of sophistication. Nevertheless, because of the evident crucial need for a molecular model for the ribosomes extensive attempts have been made in this direction. These led to many failures, but finally resulted in the ability, by us, to grow reproducibly in-vitro diffracting 3-D crystals of intact ribosomal particles. These crystals have relatively small unit cells with a reasonable degree of internal order and adequate stability in the X-ray beam. Preliminary structural information has been obtained from single crystals using synchrotron radiation as well as from 3-D image reconstruction studies. Consequently, single-crystal X-ray crystallographic studies supported by information obtained from electron microscopy are currently being carried out. The research program covered by this proposal consists of a number of points. X-ray data will be collected up to 12-15 angstroms resolution from native crystals and phases determined by both conventional and novel heavy-atom techniques. A great advantage for the production of heavy atom derivatives is the large variety of ribosomal components and the wide spectrum of materials which interact specifically with these components. In favorable cases, derivatives will be used also for an unambiguous localization of specific ribosomal components. A procedure has been developed for efficient detection of crystallization by electron microscopy. The previous 3-D image reconstruction studies will be extended, using both negatively stained 2-D crystalline sheets and positively stained thin sections of embedded 3-D crystals, for the elucidation of the overall 3-D model of the particle and incorporation of internal stain distribution. To minimize the inherent limitation of image reconstruction from thin sections, the image wil be reconstructed in different orientations. This is expected to lead to reliable results since several forms of 3-D crystal and 2-D sheets have been obtained. Results of these studies could be used for comparative structural and functional studies, and for elucidating the nature of protein-nucleic acid interactions at high salt concentrations. Structural studies will be initiated on ribosomal components from different organisms, especially from the halobacteria from the Dead Sea. In the body of this application, it is has shown that these objectives are feasible and that the extraordinary significance of the expected results justify major efforts.
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