Subunit organization and structure of lon protease

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Scanning transmission electron microscopy (STEM) has been applied to determine the subunit organization of the enzyme, lon protease, an important energy-dependent protease that is responsible for cytoplasmic protein degradation. Although the sequence of lon protease is known, its structure has not yet been determined. After first characterizing the homogeneity of the preparations by negative staining, the protein was applied to ultrathin carbon films supported on copper grids and plunge frozen into liquid ethane. Samples were freeze-dried and imaged in low dose annular dark-field STEM to provide quantitative mass maps from which the molecular weight distribution of the protein assemblies could be determined. The mass distribution was found to be bimodal revealing the existence of octamers and tetramers. In the future, attempts will be made to localize the substrate of the enzyme by averaging large numbers of low-dose images.