J. Mol. Biol. (1975) 96, 425-430 States of Aggregation of the Dahlemense Strain of Tobacco Mosaic Virus Protein and their Relation to Crystal Formation R. SPxRLINat AND A. KLua Medical Research Coun.&? Laborakny of Molecular Biology University Postgraduate Medical School Hills Road, Cambridge, England (Received 8 November 1974, and in revised forna 29 Mm~h 1975) The aggregation of the protein of the ci&lemense strain of tobacco mosaic virus has been studied by electron microscopy and ultracentrifugation. The aggregates formed are similar to those formed by the *are strain, although the particular conditions for their formation are often rather different. Helix formation by dialysis of A protein at pH 8 to acid pH is much more efficient if an intermediate step at pH 7 is introduced. The 20 S particle or two-layer disk is stable over a wide range of pH and ionic strength values. There is no tendency to form short stacks of disks at high ionic strength; instead, 30 S particles are formed that correspond to a pair of interlocked disks giving a “figure-of-eight” appearance in electron micrographs. These particles appear to be the “building blocks” of the protein crystal. 1. Introduction The dominant state of aggregation of tobacco mosaic virus protein near neut.ral pH is the 20 S disk, which consists of two layers of 17 subunits each (Durham et al., 1971, Durham & Finch, 1972). Furthermore, it is this disk that nucleates the assembly of the protein with the RNA to form the virus and also is used in the elongation of the growing rod (Butler & Klug, 1971; Butler, 1972). The 20 S disk of TMV$ protein exhibits an axial pairing interaction between its subunits, which stabilizes the disk, and a similar two-layer aggregation may be a crucial property for the assembly of other helical viruses. The pairing interaction is common to the TMY family of viruses but in the finished virus particle is exhibited especially strongly in the dahlememe strain (Caspar & Holmes, 1969). The dahlernense strain of TMY was isolated by Melchers (1949). Wittmann-Liebold & Wittmann (1963) have established that, although the vulgare and dahlenaensepro- teins are both made of 158 amino acids, the sequence of dahlemense differs from that of vulgare in 30 positions. X-ray diffraction studies of dahkmeme virus (Caspar & Holmes, 1969) have shown that it has a very similar structure to W, but there is also a periodic pairing interaction at the outside of the virus helix, which leads to a small regular deformation in the axial direction. This pairing interaction is also displayed t Present address: Chemical Physics Depertment, Weizmann Institute of Science, Rehovot, TSlYlBl. $ Abbreviation used: TMV, tobacco mosaic virus. 426