Bacteriophage T4 has a very efficient mechanism for infecting cells. The key component of this process is the baseplate, located at the end of the phage tail, which regulates the interaction of the tail fibres and the DNA ejection machine. A complex of gene product (gp) 5 (63K) and gp27 (44K), the central part of the baseplate, is required to penetrate the outer cell membrane of Escherichia coli and to disrupt the intermembrane peptidoglycan layer, promoting subsequent entry of phage DNA into the host. We present here a crystal structure of the (gp5-gp27)3 321K complex, determined to 2.9 A resolution and fitted into a cryo-electron microscopy map at 17 A resolution of the baseplate-tail tube assembly. The carboxy-terminal domain of gp5 is a triple-stranded beta-helix that forms an equilateral triangular prism, which acts as a membrane-puncturing needle. The middle lysozyme domain of gp5, situated on the periphery of the prism, serves to digest the peptidoglycan layer. The amino-terminal, antiparallel beta-barrel domain of gp5 is inserted into a cylinder formed by three gp27 monomers, which may serve as a channel for DNA ejection.

The head of bacteriophage T4 is a prolate icosahedron with one unique portal vertex to which the phage tail is attached. The three-dimensional structure of mature bacteriophage T4 head has been determined to 22-Å resolution by using cryo-electron microscopy. The T4 capsid has a hexagonal surface lattice characterized by the triangulation numbers Tend ‫؍‬ 13 laevo for the icosahedral caps and Tmid ‫؍‬ 20 for the midsection. Hexamers of the major capsid protein gene product (gp)23* and pentamers of the vertex protein gp24*, as well as the outer surface proteins highly antigenic outer capsid protein (hoc) and small outer capsid protein (soc), are clearly evident in the reconstruction. The size and shape of the gp23* hexamers are similar to the major capsid protein organization of bacteriophage HK97. The binding sites and shape of the hoc and soc proteins have been established by analysis of the soc ؊ and hoc ؊ soc ؊ T4 structures. Bacteriophage T4 is a large, tailed, double-stranded DNA (dsDNA) virus (family Myoviridae) that uses Escherichia coli as a host. The mature T4 virion, which contains Ϸ50 different proteins, consists of a prolate capsid, 172-kbp genomic DNA, and a tail with a contractile sheath terminating in a base plate to which are attached six long tail fibers. The architecture and the molecular composition of the T4 head, tail, and fibers have been characterized extensively by using a variety of techniques (1-3) leading to a structural model (4). Recent studies of the T4 components by cryo-electron microscopy (cryo-EM) and x-ray crystallography extended the structural knowledge to higher resolution (5).T4 has one of the most complex structures of any virus that has been studied. There are Ͼ2,000 protein molecules of at least five different gene products (gps) in the head alone. The molecular mass of the DNA-filled head is 194 MDa and of the capsid alone is 82 MDa (6). The T4 head assembly proceeds via a number of intermediate stages. First, a DNA-free precursor, or prohead, is assembled that is processed proteolytically. Next, the genomic DNA is packaged into the prohead in a process that requires ATP energy (1). The prohead assembly is initiated by the portal protein gp20. The prohead contains an internal core made up of the major core protein, gp22, the minor core proteins, gpalt, a serine-type protease, gp21, and other internal proteins (1). The major capsid protein, gp23, is assembled around the scaffolding core together with the minor capsid protein, gp24. After completion of prohead assembly, the inactive gp21 enzyme is converted to the active protease, which cleaves the scaffold proteins into small peptides. A 65-residue-long amino-terminal ''⌬-piece'' is also cleaved from the 56-kDa gp23 molecule, thus yielding 48.7-kDa gp23* (1) ʈ . In addition, a 2.2-kDa amino-terminal piece of the 48.4-kDa gp24 is cleaved, giving rise to gp24* during head maturation. Most of the small peptides produced by the gp21 protease are expelled from the prohead, thus providing space necessary to accommodate the geno...

Bacteriophage T4 is one of the most complex viruses. More than 40 different proteins form the mature virion, which consists of a protein shell encapsidating a 172-kbp double-stranded genomic DNA, a 'tail,' and fibers, attached to the distal end of the tail. The fibers and the tail carry the host cell recognition sensors and are required for attachment of the phage to the cell surface. The tail also serves as a channel for delivery of the phage DNA from the head into the host cell cytoplasm. The tail is attached to the unique 'portal' vertex of the head through which the phage DNA is packaged during head assembly. Similar to other phages, and also herpes viruses, the unique vertex is occupied by a dodecameric portal protein, which is involved in DNA packaging.

Gene product (gp) 24 of bacteriophage T4 forms the pentameric vertices of the capsid. Using x-ray crystallography, we found the principal domain of gp24 to have a polypeptide fold similar to that of the HK97 phage capsid protein plus an additional insertion domain. Fitting gp24 monomers into a cryo-EM density map of the mature T4 capsid suggests that the insertion domain interacts with a neighboring subunit, effecting a stabilization analogous to the covalent crosslinking in the HK97 capsid. Sequence alignment and genetic data show that the folds of gp24 and the hexamer-forming capsid protein, gp23*, are similar. Accordingly, models of gp24* pentamers, gp23* hexamers, and the whole capsid were built, based on a cryo-EM image reconstruction of the capsid. Mutations in gene 23 that affect capsid shape map to the capsomer's periphery, whereas mutations that allow gp23 to substitute for gp24 at the vertices modify the interactions between monomers within capsomers. Structural data show that capsid proteins of most tailed phages, and some eukaryotic viruses, may have evolved from a common ancestor.evolution ͉ gene product 24 ͉ major capsid protein T he protein shells of viral capsids are remarkably stable, yet dynamic structures. They have to protect the genome during its transfer between hosts, withstand the high pressure of the condensed nucleic acid, and be able to release the genome once a susceptible host has been recognized. To reconcile both stability and dynamic requirements, assembled procapsids of many viruses undergo large conformational changes during genome packaging and maturation (1).The capsid of the dsDNA tailed bacteriophage T4 is a prolate icosahedron ( Fig. 1) whose capsomers form a T end ϭ 13 laevo hexagonal lattice in the end caps and a T mid ϭ 20 lattice in the cylindrical midsection (2). The protein shell consists of the major capsid protein gene product (gp) 23*, the pentameric vertex protein gp24*, the portal protein or ''connector'' gp20, and the two accessory proteins, gp hoc (highly antigenic outer capsid protein) and gp soc (small outer capsid protein) (3), that decorate the outside of the shell. The dodecameric connector replaces a pentamer of gp24* at one of the 12 vertices and serves as a special portal for DNA packaging, tail attachment, and DNA exit (4, 5).During procapsid assembly, gp23, gp24, and gp20 form a shell around the core structure composed primarily of the scaffolding protein gp22 and assembly protease gp21 (3). The protease activates once procapsid assembly has been completed and cleaves the proteins of the core into small peptides, most of which leave the maturing procapsid, freeing space for the genome. The gp21 protease also cleaves a 65-residue-long, amino-terminal fragment from the 56-kDa gp23, generating the 48.7-kDa gp23* (3). In addition, the 10-residue, amino-terminal region of the 48.7-kDa gp24 is cleaved, giving rise to the 47.6-kDa gp24* (3). These cleavages trigger a large conformational rearrangement in the procapsid, resulting in expansion and causing the ...

The contractile tail of bacteriophage T4 undergoes major structural transitions when the virus attaches to the host cell surface. The baseplate at the distal end of the tail changes from a hexagonal to a star shape. This causes the sheath around the tail tube to contract and the tail tube to protrude from the baseplate and pierce the outer cell membrane and the cell wall before reaching the inner cell membrane for subsequent viral DNA injection. Analogously, the T4 tail can be contracted by treatment with 3 M urea. The structure of the T4 contracted tail, including the head-tail joining region, has been determined by cryo-electron microscopy to 17 A resolution. This 1200 A-long, 20 MDa structure has been interpreted in terms of multiple copies of its approximately 20 component proteins. A comparison with the metastable hexagonal baseplate of the mature virus shows that the baseplate proteins move as rigid bodies relative to each other during the structural change.

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The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal domain and the labile coiled-coil domain may be essential for the fibritin function and for the correct functioning of many other alpha-fibrous proteins.

Bacteriophage T4 and related viruses have a contractile tail that serves as an efficient mechanical device for infecting bacteria. A three-dimensional cryo-EM reconstruction of the mature T4 tail assembly at 15-A resolution shows the hexagonal dome-shaped baseplate, the extended contractile sheath, the long tail fibers attached to the baseplate and the collar formed by six whiskers that interact with the long tail fibers. Comparison with the structure of the contracted tail shows that tail contraction is associated with a substantial rearrangement of the domains within the sheath protein and results in shortening of the sheath to about one-third of its original length. During contraction, the tail tube extends beneath the baseplate by about one-half of its total length and rotates by 345 degrees , allowing it to cross the host's periplasmic space.