Radial distributions of density within macromolecular complexes determined from dark-field electron micrographs.

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Abstract
A procedure was developed for direct determination of radial distributions of density in filamentous and spheroidal particles by analyzing dark-field scanning transmission electron micrographs of unstained freeze-dried specimens. Unlike EM methods based on staining or shadowing with heavy atoms, this approach can be used to probe the internal structure of macromolecular complexes. An an experimental proving ground, the procedure was applied to tobacco mosaic virus (TMV) and to RNA-free helical polymers of TMV coat protein. Both structures are found to project outermost diameters of 17.6 .+-. 0.4 nm, to have empty axial holes .simeq. 3.5 nm in diameter, and to have density peaks at radii of 2.5 .+-. 0.5 and 6.7 .+-. 0.3 nm. Thus visualized, the only significant difference between them is the presence in the virion of an additional density peak at 4.1 .+-. 0.5 nm contributed by its internalized RNA molecule. The procedure was used to monitor the structural expression of radiation damage in the low electron dose regime prior to the onset of significant mass loss. Changes in the radial density profiles are detected at average dose as low as .simeq. 400 electrons per nm2: the trend is for the internal structure of these particles to fuse toward a state of uniform density, although the values of their outermost diameters remain unaffected.