STRUCTURAL AND ELEMENTAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

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The goal of this project is to characterize in vitro the shape, molecular weight distribution and elemental composition of specific individual macromolecules and macromolecular assemblies, with emphasis on components of the cytoskeleton. This project depends on a unique instrument -- a field-emission scanning transmission electron microscope (STEM) -- equipped with dark-field detectors (for mapping the molecular weight distribution of molecules at a spatial resolution of 2 nm) and a parallel electron energy loss (EELS) spectrometer (for detecting physiologically relevant differences in phosphorylation states at a resolution of 10-20 nm). Applying this method to rapidly-frozen, freeze-dried neurofilaments isolated from the squid giant axon, we have been able to derive a novel structural model for the arrangement of heavy chains (highly phosphorylated) and light chains (weakly phosphorylated) in native axonal neuro-filaments. The STEM measurements give a neurofilament mass-per- length of 22.7 plus minus 0.8 kDa/nm which implies only eight coiled-coli dimers per cross-section. The EELS results indicate four heavy chains per cross section, each containing approximately 50 to 60 phosphates; this is essentially the maximum predicted by the amino acid sequence. We have also begun to exploit an energy-filtering electron microscope (EFTEM) as a complementary approach for the analysis of cellular phosphorus. EFTEM has been previously used for mapping of phosphorus distributions in molecular assemblies, but at high electron dose, which leads to specimen damage. Recent experiments now show that it is possible to obtain quantitative, element-specific images of directly frozen thin films of biological specimens at low dose and therefore potentially at high resolution. Thus, we have obtained phosphorus-specific, low-dose images of herpes simplex virus particles with and without their DNA cores. The difference between these images reveals a statistically significant quantitative map of the distribution of the DNA within the virus. These results suggest that it may be possible to carry out such experiments even in fully hydrated specimens.