Arrangement of protein subunits and the distribution of nucleic acid in turnip yellow mosaic virus: I. X-ray diffraction studies

Abstract

The protein shell of turnip yellow mosaic virus is known from earlier X-ray work to possess icosahedral symmetry, and the surface structure of 32 knobs (“morphological units”) deduced from electron microscope observations is in accord with this. The present paper describes an X-ray investigation to determine the arrangement of the ultimate protein subunits (“structure units”) and the distribution of the RNA within the virus particle. X-Ray data on both the virus and the nucleic acid-free “top component” have been obtained systematically out to spacings of about 20 A. The crystal structure of newly grown crystals of fresh preparations of both virus and top component is almost always the diamond-type lattice (with 8 particles per unit cell) originally found by Coslett & Markham (1948) and Bernai & Carlisle (1948) . The double-diamond lattice with 16 particles per cell observed by Klug, Finch & Franklin (1957) has not been found again, although intermediate crystal forms with more than 8 but less than 16 particles per cell have been identified. The variability in crystal structure accounts for the erroneous conclusion drawn from the earlier X-ray work that the over-all (low-resolution) symmetry of the ordering of the RNA within the virus particle was lower than that of the protein. Photographs from crystals with a well-defined lattice show that the gross distribution of the RNA has the same icosahedral symmetry as the protein to the limit of resolution of the present study (20 A). The large complex cubic cell of turnip yellow mosaic virus has the property that certain hypotheses about the distribution of matter in the particle can be tested without solving the structure directly. (As shown in the Appendix, certain classes of X-ray reflections correspond separately to the real and imaginary parts of the transform of a single particle, and others are a measure of the departures from spherical symmetry.) Moreover, high-angle X-ray photographs (spacings The theory of Caspar & Klug (1962) provides a way of enumerating and describing the possible types of arrangements of structure units in icosahedral shells, and permits systematic calculations to be made on model structures for comparison with the X-ray patterns. The physico-chemical data on the virus are discussed and point to the icosahedral surface lattice T = 3 corresponding to 180 protein subunits. The comparison of the calculations with the X-ray data on top-component particles gives good agreement for a model which has 180 scattering centres lying at a radius of about 145 A with the spherical co-ordinates shown in Fig. 3(c). These points are identified with the protuberances of the protein structure units at the surface of the particle. The comparison of the calculations with the X-ray data on the virus gives good agreement for a model with 32 scattering centres lying at a radius of about 125 A with the spherical co-ordinates shown in Fig. 3(d). This fluctuation in density so deep within the virus particle is attributed to the presence of the RNA, which has a considerably greater scattering power than the protein. Corifirmation of the deduced distribution of RNA has been obtained by two methods: (i) staining with uranyl acetate which enhances the RNA scattering; (ii) matching the protein scattering by workingjwdth crystals in high salt concentration. The results on the distribution of RNA and protein within the virus particle are summarized by the diagram in Fig. 4(a) and the 3-dimensional model in Plate X. A significant proportion of the RNA is deeply embedded within the protein shell, and the mode of winding of the single RNA chain must be such that large segments of it are intimately associated with the rings of 6 and 5 protein structure units which make up the protein shell. It is the presence of the RNA in and about these positions that enhances the appearance of 32 morphological units in the electron micrographs (see also paper II of this series, Finch & Klug, 1966 ).