Structure of tomato bushy stunt virus: I. The spherically averaged electron density

Abstract

The radial electron-density distribution of tomato bushy stunt virus has been determined at low resolution by X-ray methods. Three experimental techniques have been used jointly to measure the spherically averaged Fourier transform of the particle in media of different electron density: (1) small-angle X-ray scattering from the virus in solution (counter recording); (2) small- and moderate- angle X-ray scattering from gels of the virus (photographic recording); and (3) single-crystal X-ray diffraction. The continuous transform corresponding to five different background densities has been obtained from the measurements with solutions and gels. The electron densities range from 0.343 e A ̊ 3 to 0.408 e A ̊ 3 . The single-crystal diffraction experiments show that no structural change or change in solvent binding occurs on transfer of the virus particle from one of these media to another, and that ammonium sulfate, sodium sulfate and sucrose can be used interchangeably to alter the solvent density. The single-crystal data extend to sufficiently wide angles that intensities from crystals in different media can be scaled to each other, since the background density has little effect on the diffraction pattern at spacings smaller than 20 Å. The continuous transforms can therefore be compared by scaling them to the corresponding crystal diffraction patterns. Density measurements by flotation in salt or sucrose solutions show that there is approximately 0.4 g water bound per gram virus in crystal and gels. This figure is also consistent with measurements of the intensity of the central maxima of X-ray scattering patterns from bushy stunt virus in solutions of different electron density. The bound water, defined as water from which small solutes are excluded, is associated with both protein and RNA. The net electron density in the tomato bushy stunt virus particle can be calculated, as a function of radius, by Fourier transformation of the appropriately phased scattering amplitudes. A comparison of the radial density distribution in different solvents indicates that matter of high intrinsic density, interpreted as RNA, is concentrated at a radius of about 110 Å. Both inside and outside this shell, the density of the solvent-impenetrable regions has values characteristic of hydrated protein. Fourier inversion of the salt-difference transform yields a measure of the volume fraction occupied by solvent at any radius. The fraction of the volume available to solvent is greater in the RNA region than at radii at which the internal and external protein layers are most densely packed. The tomato bushy stunt virus particle thus appears to have a protein “core” and a protein “coat”, perhaps built from the same kind of protein subunits. The RNA lies between these two shells. Some of the implications of this model—first proposed by Klug and co-workers are briefly discussed.