My research will attempt to answer the question "How much do prokaryotic cells resemble bags of enzymes or well-ordered arrays?" Cryoelectron tomography will be used to pursue the following aims: (1) Determine the ultrastructure of the E. coli cytoskeleton. Recently-discovered, actin-like filaments like MreB were found to be critical for maintaining cell-shape, cell division and genome segregation, and shall be located in tomograms. (2) Determine the structure of the actin-like ParM bundle responsible for segregating the R1 plasmid. A wild type and hyperfilamenting form of ParM shall be transformed into E. coli, in order to see the arrangement of strands within the cell. (3) Determine the structure of the megadalton-sized, E. coli pyruvate dehydrogenase (PDMC) and 2-oxoglutarate dehydrogenase (ODMC) complexes. These two large complexes and the ribosome should be detectable in E. coli tomograms. Preliminary results show that the complex is inherently flexible, which differs from the theoretical model. (4) Determine the intracellular spatial organization of the largest E. coli macromolecular complexes - PDMC, ODMC and the ribosome - with automated searches in tomograms using the program Oscar on the department's supercomputer. [unreadable] [unreadable]