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Structural changes of bacteriophage phi29 upon DNA packaging and release - PubMed

  • ️Sun Jan 01 2006

Comparative Study

. 2006 Nov 1;25(21):5229-39.

doi: 10.1038/sj.emboj.7601386. Epub 2006 Oct 19.

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Comparative Study

Structural changes of bacteriophage phi29 upon DNA packaging and release

Ye Xiang et al. EMBO J. 2006.

Abstract

Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage phi29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the phi29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5' ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.

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Figures

Figure 1
Figure 1

Assembly pathway of φ29. The empty prohead is assembled from the scaffolding protein (gp7), the major capsid protein (gp8), the head–tail connector (gp10), the head fibers (gp8.5), and pRNA. Packaging of DNA–gp3 requires the ATPase gp16 and ATP. The scaffolding protein is lost during DNA packaging. When the DNA–gp3 has been packaged, the pRNA and gp16 components of the packaging machine come off the packaged prohead and are replaced sequentially by the lower collar (gp11 and gp13) and knob (gp9), which, together with the appendages (gp12*), make up the tail. During cell infection, the DNA–gp3 is ejected through the tail, a process that can be mimicked in vitro by treatment with NaClO4.

Figure 2
Figure 2

The φ29 structure during the virus' life cycle. Comparison of the cryoEM maps of (A) full (blue) with emptied (red) particles and (B) empty prohead (green) with emptied virion (purple) particles. The width-to-length ratios are the same for all three particles. The maps shown in (A) and (B) are 15 Å-thick slabs showing only the head and proximal parts of the tail. Contours are shown in 2σ intervals. (C) Organization of DNA in mature particles. At least three layers of the packaged genome are resolved in the central cross-section. High densities are white, low densities are black. Various structural components of the phage are labeled, including the dominant, well-defined densities I, II, and III. Horizontal lines designate the approximate boundaries of the different tail components.

Figure 3
Figure 3

The DNA strong density I (see Figure 2C) near the portal vertex. (A) Stereo view of circular DNA (green) near the connector density (lilac). The capsid, head fibers, lower collar, and appendages are shown in lime green. (B) Side view of circular DNA (green) in the vicinity of the fitted head–tail connector structure (red). The crystal structure of the dodecameric connector is shown as a ribbon diagram.

Figure 4
Figure 4

Changes in the connector structure upon phage maturation. (A) Fit of the modified gp3 crystal structure (ribbon representation in orange) into the strong density II (see Figure 2C) in the center of the connector as visualized in the asymmetric cryoEM reconstruction. The structure of the head–tail connector (Cα backbone trace, red) is shown fitted into the density (blue) of the mature phage contoured at 2σ intervals. The DNA density is shown in green. (B) Fit of the modified gp3 crystal structure (orange) into strong density III (see Figure 2C) in the tail's lower collar as visualized in the asymmetric reconstruction. (C) Fit of the connector crystal structure (Cα trace in red) into the cryoEM density of the five-fold averaged reconstruction of the prohead and (D) the cryoEM density of the asymmetric reconstruction of the emptied particles. The narrow end of the connector would have to increase its radius in order to fit into the density (blue) of the emptied particle.

Figure 5
Figure 5

Structure of appendages labeled 1–12. Surface-shaded views showing (A) an angled side view contoured at 4.5σ and (B) a top view contoured at 3σ. Appendages 1 and 6 are in the ‘up' position, whereas the other appendages are in the ‘down' position. (C) Diagram showing the relationship between the position of the appendages and the position of the head fibers. The appendages in the ‘up' position are shown as green ellipses, whereas those in the ‘down' position are shown as green circles. The lower tier of five fibers (A1–A5) is shown in pink. The more distant upper layer of 10 fibers (B1–B10) is shown in yellow. Note that appendages in the ‘up' position are least hindered by the head fibers.

Figure 6
Figure 6

Sequence and structural alignment of φ29 gp12, plant RGase, and the P22 tailspike protein. The predicted secondary structure of gp12, and the observed secondary structures of RGase and the P22 tailspike are shown above the alignments. Conserved residues are boxed. Residues involved in substrate binding and catalysis in RGase and the P22 tailspike are shown in white on a black background. Completely conserved residues are shown in white on a gray background. The program JPred was used for making the secondary structure prediction of gp12 (Cuff and Barton, 2000).

Figure 7
Figure 7

Stereo diagram showing the fit of the P22 tailspike structure into the cryoEM density map as determined by the asymmetric reconstruction for the mature φ29 phage. The trimeric P22 tailspikes are shown as ribbon diagrams using red, green, and blue for the three different monomers. The cryoEM density is shown in gray.

Figure 8
Figure 8

Comparison of the knob and lower collar cryoEM density of the mature and DNA-emptied particles. The DNA-emptied particles have an additional cone-shaped density at their distal end as seen in (A) a surface shaded view and (B) a cross-section of the cryoEM densities. The mass of the cone-shaped density appears to form the end of the knob in the mature virus.

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