Bubblegrams Reveal the Inner Body of Bacteriophage ϕKZ

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Science  13 Jan 2012:
Vol. 335, Issue 6065, pp. 182
DOI: 10.1126/science.1214120


Dense packing of macromolecules in cellular compartments and higher-order assemblies makes it difficult to pick out even quite large components in electron micrographs, despite nominally high resolution. Immunogold labeling and histochemical procedures offer ways to map certain components but are limited in their applicability. Here, we present a differential mapping procedure, based on the physical principle of protein’s greater sensitivity to radiation damage compared with that of nucleic acid.

ϕKZ is a large and complex virus that infects the Gram-negative bacterium Pseudomonas aeruginosa and has long-term potential for phage therapy against this pathogen. The virion has a long contractile tail and a large icosahedral capsid containing densely packed DNA (280 kb) (1). Observations of disrupted virions (2) have shown that it also contains a cylindrical structure called the inner body. However, the inner body is invisible in conventional cryogenic electron micrographs of intact virions because it cannot be distinguished from the surrounding DNA (Fig. 1A).

Fig. 1

Cryoelectron micrographs of purified ϕKZ virions: (A) initial low-dose exposure; (B) subsequent exposure of the same field, with bubbling in radiation-damaged virions. (C) Three-dimensional reconstruction of the ϕKZ capsid viewed along the axis of fivefold symmetry that passes through the portal. The capsid has T = 27 icosahedral symmetry (4). The centers of all hexons on one icosahedral facet are marked with red dots. Hexons in the ring of five, on one of which the inner body (in magenta) is anchored, are colored green. The symmetry-related hexons on the other side of the capsid are in pale green in the bottom image. Each of these hexons lies on a facet edge, connecting two vertices. The central axis of the inner body passes through the center of the capsid. (D) Central section of the ϕKZ head sampled in the plane in which the inner body axis lies. This view illustrates the tilt of the inner body relative to the portal axis and the offsetting of its point of contact with the capsid from the portal vertex. (E) Multitiered structure of the inner body shown in surface rendering (left, magenta) and central gray-scale section (right). The structures in (C) and (E) were sixfold rotationally averaged (justified by the angular power spectra analysis in fig. S3); the structure in (D) was not.

It has been found, serendipitously, that the inner body is exceptionally sensitive to radiation damage and explodes into bubbles of gaseous radiation products at electron doses that leave most protein complexes, including the surrounding capsid, only slightly blurred (Fig. 1B). We were able to determine its structure by using these “bubblegrams” to locate the inner body in individual nucleocapsids; then, knowing these locations and orientations, we calculated a three-dimensional reconstruction of the inner body from previously recorded, low-dose micrographs depicting the same virions in a relatively undamaged state.

The inner body is ~24 nm wide and ~105 nm long and tilted at ~22° relative to the portal axis (Fig. 1, C and D). It consists of multiple stacked tiers (Fig. 1E), and some regions have evident sixfold symmetry, as confirmed by their angular power spectra [supporting online material (SOM)]. The two ends, which are structurally distinct, are anchored on opposing hexons on either side of the capsid. The inner body has several major proteins and a number of minor proteins (3). The volume of the inner body as calculated here suggests a total mass of about 15 MD. The shape and position of the inner body suggest that it plays a role of organizer in the DNA packaging process. Consistent with this assignment, its tilt relative to the portal axis matches that of the ϕKZ DNA spool (4). The inner body is dismantled when the DNA is ejected from the capsid during infection (2). Inner body proteins are also likely injected into the host cell, based on the precedent of phage T7 (5), which also has a multitiered internal protein structure (6).

Bubbling is the end point to damage induced by electron irradiation of ice-embedded proteins (7). Although a detailed understanding of the radiation chemistry is lacking, this phenomenon appears to represent the formation of gaseous hydrogen-containing bubbles at high pressure (8). Why do inner body proteins bubble at relatively low electron doses (~50 electrons per Å2 in a 0.5-s exposure)? Because there is no evidence to suggest that inner body proteins are chemically distinct from other proteins, we posit that they have a propensity to bubble because they are embedded in DNA. This impedes the diffusion of radiation products from their site of origin and promotes their build-up to concentrations at which bubbles nucleate. Support for this interpretation comes from the absence of bubbling in DNA-free particles containing the same inner body proteins. We suggest that bubblegram imaging may be productively applied to map proteins in other DNA-rich contexts such as cryosections of the cell nucleus.

Supporting Online Material

Materials and Methods

Figs. S1 to S4

References (914)

References and Notes

  1. Acknowledgments: This work was supported by the Intramural Research program of NIAMS and by NIH grant AI11676 to L.W.B. The structure has been deposited at the Electron Microscopy Data Bank, reference number 10395, accession code EMD-1996.
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