Cell, Vol. 7, 191-203, February 1976, Copyright 0 1976 by MIT Correlation between Structural Transformation and Cleavage of the Major Head Protein of T4 Bacteriophage U. K. Laemmli Department of Biochemical Sciences Princeton University Princeton, New Jersey, 08540 L. A. Amos and A. Klug MRC Laboratory of Molecular Biology Hills Road Cambridge, England Summary We have studied the maturation of T4 polyheads, the aberrant tubular structures related to the cap- sid of T4 bacteriophage. Conditions have been found under which more than 95% of the major head protein (P23) undergoes the same cleavage that occurs during development of the normal cap- sid. The concomitant structural changes in the poly heads have been followed using electron micro- scope image filtering techniques. As a result of the cleavage, a radical transformation of the hexagonal lattice occurs, involving a 1 O-l 5% expansion in the lattice dimensions. However, a metastable interme- diate state similar to the uncleaved structure has been observed immediately after cleavage of the protein subunits. Some kind of additional physical stimulus seems to be required to trigger the major structural change, which appears to be highly cooperative. Introduction Considerable progress has been made in elucidat- ing the pathway which leads to the mature head of the bacteriophage T4. The major feature of this pathway is that empty heads are first formed and then the DNA is pulled into them. The first particle on the pathway is called prohead I: it contains the precursor protein P23, and possibly the protein P22 and the internal protein IPIII (Laemmli and Favre, 1973). The prohead I appears to contain no DNA, is very fragile in cell lysates, and is converted to a more stable particle prohead II, as a result of the cleavage of the major capsid protein P23 (mo- lecular weight = 55,000 daltons) to P23* (molecu- lar weight = 45,000 daltons) (Laemmli and Favre, 1973; Bijlenga, van der Broek, and Kellenberger, 1974). The proheads I and II contain no DNA and appear not to be attached to the replicative DNA complex. DNA is packaged during the conversion of prohead II to prohead III and during the succeeding conver- sion of prohead III to the mature head. This DNA packaging event is accompanied by the cleavage of the core protein P22 to small fragments (Laemmli and Favre, 1973; Laemmli, Teaff, and D’Ambrosia, 1974a). The protein P22 is the major component (Laemmli and Quittner, 1974) of the internal core seen in the precursor particles and the various aberrant head-related structures (Kellenberger, Ei- serling, and Boy de La Tour, 1968; Simon, 1972). The prohead I particles found in pulse-labeled wild-type infected cells are thought to be essentially identical to the r particles (see Laemmli et al., 1974). The latter particles accumulate in cells infected with a phage carrying a mutation in gene 21 or 24 (Ep- stein et al., 1963; Kellenberger et al., 1968; Laemmli et al., 1970). Recent experiments showed that 7 particles formed at nonpermissive temperatures in cells infected with a temperature-sensitive mutant phage in gene 24 can be converted into active phage if the product of gene 24 is activated by tem- perature shift (Bijlenga et al., 1974). Thus gene 24 appears to control the conversion of prohead I (7 particle) to prohead II. A puzzling feature of the pathway is the observation that the T particles are about IO-20% smaller in width and length than the mature head (Kellenberger et al., 1968). One would expect, therefore, that the prohead I particles have to be enlarged either by the addition of new sub- units or by a rearrangement of the surface subunits into a surface lattice of expanded dimension during this maturation. In this paper we present direct evi- dence for a structural rearrangement of the surface lattice induced by the cleavage of P23 to P23”. The structural rearrangement has been studied with the help of the tubular aberrant polyhead of phage T4 produced in mutants of gene 20 (Epstein et al., 1963). This particle has many times the length of the normal phage head and has proved very suit- able for analysis of the subunit structure by optical diffraction and image reconstruction (DeRosier and Klug, 1972; Yanagida, DeRosier, and Klug, 1972). The surface structure of polyheads has been stud- ied extensively and is based upon a plane hexago- nal lattice (symmetry p6) rolled into a cylindrical sur- face (Klug and Berger, 1964; Kellenberger and Boy de la Tour, 1965). Closer analysis showed that fresh lysates contained predominantly polyheads with a coarse structure, while smooth-surfaced polyheads predominated in aged lysates (DeRosier and Klug 1972). The appearance of the smooth polyheads in negatively stained preparations is similar to that of the mature phage head. It was therefore suggested that the different appearances in the filtered images of polyheads might mimic the different stages of normal head maturation (Yanagida et al., 1970), al- though at that time it could not be proved that the structural changes were correlated with the chemi- cal processing of the proteins. Since then, Laemmli and Quittner (1974) have found conditions to control the cleavage of the poly- heads in vitro, and it has been possible to follow the chemical and structural changes in parallel. In