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The form of the bacteriophage T4 prehead is described by its icosahedral symmetry, its diameter, and its length. We show how each oftheseparameters is regulated during prehead formation and ascribe specific form-determining functions to the prehead proteins. The major protein of the head shell can assemble in several different forms. The structure produced in vivo depends on the rate of synthesis of the major protein relative to the rates of synthesis of minor shell proteins and the major core protein. From our observations, we propose a model for form determination of the prehead and suggest a pathway for the evolution of its prolate shape.The head of bacteriophage T4 has been extensively studied as a model for discovering mechanisms of form determination of biological structures. It is a prolate icosahedron of triangulation number (T) = 13 (1, 2) elongated on its 5-fold axis of symmetry (3), with a length-to-width ratio of 1.37 (4). It is assembled on the Escherichia colh inner membrane as a core-containing prehead (5), which is matured to the finished head by a series of reactions that includes limited proteolysis of capsid and core proteins, expansion of the prehead shell lattice, and packaging of the phage DNA (6). The relatively large size and the prolate shape of the T4 head suggest that the prehead [in which the form is already determined (4)] cannot be produced by simple self-assembly of the shell protein, as is possible with small regular icosahedral viruses (7,8).A number of mechanisms have been proposed (9, 10) to account for form determination in the assembly of large or anisometric virus shells or other structures in which simple selfassembly of identical subunits is not possible (11). They include regulation by a genetically defined "measuring rod" or template, a vernier, and accumulating strain with the addition of subunits. Of these, only the "measuring rod" model, (the determination of the length of tobacco mosaic virus by its RNA) has been experimentally demonstrated (12,13). All of these models depend strongly on specific interactions between the polymerizing subunits, but Wagenknecht and Bloomfield (14) have shown theoretically how the length of linear aggregates might also depend on the concentration of the polymerizing subunits.Recent observations on the role of minor proteins in prehead formation, and on changes in prehead length that depend on subunit concentrations, suggest that kinetic factors as well as specific protein-protein interactions regulate the form of the T4 prehead.The form of the T4 prehead is described by its symmetry (icosahedral), its width, and its length (extension along the 5-fold axis of symmetry). We account for these in the following way. The symmetry is determined by formation of a 5-fold symmetric initiation complex that directs assembly of the shell into closed structures instead of open tubes. The width is determined by the intrinsic curvature of the shell and the diameter of the core that it must enclose. The length can be varied. It is regulated by t...

The bacteriophage T4 capsid contains a number of minor proteins that are required for head assembly but whose detailed function and position in the head are unknown. We have found that by systematically varying the conditions of extraction, some of these minor proteins can be removed while the main capsid structure is left substantially intact. Electron microscopic examination of the residual capsids showed that the extraction ofthe product of gene 20 is correlated with the loss of a plug that distinguishes one vertex position (presumably the tail attachment site) from the others. Extraction of the product of gene 24 is correlated with the loss of the other 11 (nonproximal) vertexes of the capsid. We further show that antibody to P24 binds specifically to the nonproximal vertexes of both T4 preheads and T4 phages. On the basis of our findings, we suggest that P20 is located at or near the tail attachment site ofthe capsid, whereas P24 forms the 11 nonproximal vertexes of preheads and P24* forms the nonproximal vertexes of the mature head.

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ABSTRACr Fab fragments prepared from antisera directed against purified bacteriophage T4 structural proteins-and head-related structures were used to label proteins on the surface of T-even giant phage capsids. Optically filtered electron micrographs of the Fablabeled capsids reveal both the location of specific proteins within the capsomeres and differing conformational states of the protein subunits. We describe parameters affecting the utility of this technique for the study of molecular organization and protein conformation in periodic biological structures. Many biological structures, including virus shells, contractile filaments, microtubules, and parts of bacterial cell walls, are built up as ordered arrays of one or several species of protein subunits. Electron microscopy of negatively stained specimens followed by image processing of the electron micrographs has proved to be a useful tool for establishing the supramolecular organization of these asetblies (1). The most common wayol localizing individual protwein subunits within their repeating unit has been to compare th: filiered images of related structures that differ in the*otein cporsition. These are usually obtained either from mutants that cannot synthesize one or more of the constituents (2, 3), by differential dissociationof the structures (4), or by in vitro complementation of the defrcient structures with the proteins they are lacking (2,(4)(5)(6) phage T4 capsids we have been able to confirm the localization (2, 4) of these proteins within the capsomere. Furthermore, this technique has allowed us to demonstrate an induced conformational change on the binding of one of these proteins to the basic capsid matrix.MATERIALS AND METHODS Antigens and Antisera. The T4 outer capsid proteins hoc and soc were purified as described by Ishii and Yanagida (2). T4 coarse polyheads (13) composed only of the product of gene 23 (P23) obtained from a mutant in gene 20 were purified by differential centrifugation. Gene 23ts aberrant preheads containing most of the T4 head proteins including hoc and soc (L. Onorato, unpublished data) were purified by two successive sucrose gradients (14). A 200-to 500-,gg sample of each antigen was injected in Freund's complete adjuvant into the hind footpads of rabbits. Animals were bled twice weekly, starting 4 weeks after injection, and serum from several bleedings was pooled for the preparation of IgG and Fab fragments. The production of high-titer soc antiserum required bimonthly intravenous boosting with 50 jig of the purified antigen. The sera were~characterized on Ouchterlony plates and by immunoreplicate electrophoresis (15). The anti-hoc serum gave no reaction with T2 phage proteins, but reacted strongly with hoc and with another T4 protein, possibly derived from hoc. Antisoc serum reacted only with soc protein. Antiserum to 23ts aberrant preheads reacted strongly with hoc, weakly with P23 and P24, and gave no detectable precipitin reaction with soc or the proteolytically processed form of P23 found in the mature phage...