Macromolecular structures of high complexity are being studied using a high resolution electron microscope with tilt/rotation capability and novel methods of image analysis. These methods allow reproducible structural information to be extracted from images of non crystalline specimens. Images of single molecules in characteristic views are aligned, objectively sorted by multivariate classification and averaged to yield statistically significant projections. (i) The three-dimensional structure of two ribosomal particles (50S subunit and 70S monosome) will be reconstructed from projections so obtained. The 3-D model of the 30S subunit already reconstructed will be further refined. The use of unstained frozen, hydrated specimen and low dose techniques will be explored to maximize the information yield. (ii) The method of localizing ligands with high accuracy (10A) will be further developed on ribosomes (t-RNA-30S complex and antibody labeling) and hemocyanins. Specifically, this method will be used to map the location of constituent proteins relative to the 3-D models of the 30S ribosomal subunit and the 48-mer of Limulus polyphemus hemocyanin. (iii) Independently reconstructed 30S, 50S, and 70S ribosomal particles will be reconciled by computer fitting to obtain a precise model for the relative arrangement of the large and small subunit in the ribosome. (iv) Inter- and intra-kingdom comparisons of the small subunit will be made by averaging images showing the particle in equivalent views.
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