Electron Microscopy of Negatively Stained and Frozen-Hydrated Bacteriophage Φ29

Abstract

Bacteriophage Φ29 is a complex-shaped, double-stranded DNA virus that infects Bacillus subtilis . Mature Φ29 particles contain six major structural proteins which give rise to several distinctive features: a prolate-shaped head (gp8), fibers (gp8.5) which radiate from the head, a connector-collar region (gp10 and gp11), collar appendages (gp12*) and a tail (gp9). Although much is known about Φ29 biochemistry, genetics and assembly, relatively little is known about its detailed structure. Three-dimensional reconstructions of the collar demonstrated that it contains two structurally distinct features: an upper portion (proximal to the head) with 12-fold axial symmetry (gp10) and a lower protion with 6-fold symmetry (possibly both gp10 and gp11). The capsid is believed to be organized with two apical regions with trimeric aggregates of gp8 dimers arranged on a T=l icosahedral lattice separated by 10 additional trimers in the equatorial region. Φ29 proheads are assembly intermediates that contain only gp8, gp8.5, gp10 and the scaffolding protein (gp7) which is released upon DNA encapsidation. We have imaged Φ29 with conventional negative-staining and the recently developed cryo-microscopy techniques with the goal of examining the structural basis for Φ29 morphogenesis.Vitrified Φ29 proheads display distinct outlines with one end pointed and the other somewhat flattened and they often orient with their long axes in the plane of the vitreous water (Fig. 1). Occasional end-on views with a circular profile are also observed. Proheads show a tendency to associate in pairs with the closest contact between the flattened ends (Fig. 1, arrow-head). Longer morphological variants are also occassionally observed (Fig. 1, arrow). The morphology of proheads is dramatically distorted in negative-stain preparations indicating significantly poorer preservation using this technique (Fig. 2). Proheads are also permeable to stain.