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The virions of tailed bacteriophages and the evolutionarily related herpesviruses contain, in addition to highly condensed DNA, substantial quantities of internal proteins. These proteins (“ejection proteins”) have roles in scaffolding, maturational proteolysis, and cell-to-cell delivery. Whereas capsids are amenable to analysis at high resolution by cryo-electron microscopy, internal proteins have proved difficult to localize. In this study, we investigated the distribution of internal proteins in T4 by bubblegram imaging. Prior work has shown that at suitably high electron doses, radiation damage generates bubbles of hydrogen gas in nucleoprotein specimens. Using DNA origami as a test specimen, we show that DNA does not bubble under these conditions, it follows that bubbles represent markers for proteins. The interior of the prolate T4 head, ~1000 Å long by ~750 Å wide, has a bubble-free zone that is ~100–110 Å thick, underlying the capsid shell from which proteins are excluded by highly ordered DNA. Inside this zone, which is plausibly occupied by ~4 layers of coaxial spool, bubbles are generated at random locations in a disordered ensemble of internal proteins and the remainder of the genome.

&ldquo, Giant&rdquo, phages have genomes of &gt, 200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them &ldquo, mottled capsids&rdquo, Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a &ldquo, twisted hook&rdquo, conformation. Large patches of a proteinaceous tetragonal lattice with a 67 &Aring, repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.