Assembly of a tailed bacterial virus and its genome release studied in three dimensions

Abstract

We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage phi29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.

Figure 1. Simplified Bacteriophage f29 Assembly Pathway

Figure 2. Cryo-Electron Microscopy and Three-Dimensional Image Reconstructions of Bacteriophage ϕ29 Particles

Micrographs of ϕ29 particles embedded in vitrified ice (A–E; scaled to the same magnification), and surface-shaded views of the corresponding 3D reconstructions (F–J,M–O). (A and F) Fiberless, isometric particles. (B and G) Fibered, isometric particles. (C and H) Proheads. (D and I) Mature phage. (E and J) Emptied phage. Prolate particles generally orient with the long axis nearly parallel to the vitreous ice surface (C–E). Fiberless (F) and fibered (G) isometric particles show protruding domains of gp8 colored red, green, and blue to highlight the three quasi-equivalent subunits in the T=3 capsid, and four fibers are highlighted in yellow. (K) T=3 triangulation net consisting of 60 triangles that define the lattice organization of the isometric structures in (F) and (G). Yellow dots mark the location of several of the 60 quasi-3-fold axes and hence the location of the fibers. The sites of pentamers and hexamers are identified by white and red symbols, respectively. (L) Q=5 triangulation net with the bottom vertex missing showing the lattice organization of the prolate structures in (H–J). Fifty-five triangles contain quasi-3-fold axes (yellow dots), whereas the remaining 20 (light blue), at the equator, have local 3-fold symmetry. Pink symbols mark the sites of equatorial hexamers. (M–O) Same as (H–J), with the front halves removed to reveal internal features such as the thin capsid wall, the dsDNA, and the structures of the connector and tail assemblies. Table 2 lists details about the microscopy and data analysis.

Figure 3. Schematic Diagram of an Emptied ϕ29 Phage

Distances are given in Å. Numbers in parentheses correspond to the mature phage. The 420 Å dimension of the prolate head refers to the external diameter.