DNA packaging intermediates of bacteriophage φX174 Leodevico L Ilag, Norman H Olson, Terje Dokland, Cynthia L Music, R.Holland Cheng, Zorina Bowen, Robert McKenna, Michael G Rossmann, Timothy S Baker, Nino L Incardona  Structure  Volume 3, Issue 4, Pages 353-363 (April 1995) DOI: 10.1016/S0969-2126(01)00167-8

Figure 1 Assembly pathway of φX174 based on Hayashi et al. [4]. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 2 The F (pale blue), G (red) and J (green) proteins in the virion as determined from the refined, 3 å resolution, X-ray crystallographic structure [16] viewed down an icosahedral two-fold axis. Two-fold, three-fold and five-fold axes (indicated by an ellipse, two triangles and a pentagon, respectively) surrounding an icosahedral asymmetric unit are also shown. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 3 Cryo-electron micrographs and three-dimensional reconstructions of φX174 assembly particles. Images of frozen-hydrated particles (top row), shaded surface representations (center row) and cross-sections (bottom row) of each reconstruction are shown of the procapsid (a,d,g), the provirion (b,e,h) and the virion (c,f,i). The small particles in (a) and (b) are degraded procapsids and provirions that have lost the D scaffolding protein and are similar in structure to virions. The shaded surface representations and cross-sections are viewed down a two-fold axis. Spikes are observed along the periphery of the virions (f) and (i), but not on the procapsids, (d) and (g), or provirions, (e) and (h). The magnification bar corresponds to 1000 å (a–c) and 200 å (d–i). Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 4 SDS-PAGE analysis of φX174 particles. A 20% polyacrylamide gel with 0.5% cross-linking agent was run in 0.75 M Tris-HCl (pH 9.3) buffer with 10% glycerol. The gel was loaded with virions in lane a, procapsids in lane b and provirions in lane c. Protein B migrates abnormally slowly under these conditions in an SDS gel [6]. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 5 (a) Fit of the X-ray crystallographically determined atomic structure to the cryo-EM density of 70S particles (partially empty virions). An icosahedral three-fold axis runs vertically through the center of the figure. Notice the small spike made by helices α4 on the surface of the virus at the three-fold axis. (b) Comparison of the procapsid (blue), provirion (green) and virion (red) cryo-EM densities viewed with the icosahedral five-fold axis running vertically through the center of the figure. The big spike of the virion on the five-fold axis is produced by G protein pentamers. The pentamer is condensed radially inwards by ∼11 å in the virion (red) relative to the assembly intermediates. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 5 (a) Fit of the X-ray crystallographically determined atomic structure to the cryo-EM density of 70S particles (partially empty virions). An icosahedral three-fold axis runs vertically through the center of the figure. Notice the small spike made by helices α4 on the surface of the virus at the three-fold axis. (b) Comparison of the procapsid (blue), provirion (green) and virion (red) cryo-EM densities viewed with the icosahedral five-fold axis running vertically through the center of the figure. The big spike of the virion on the five-fold axis is produced by G protein pentamers. The pentamer is condensed radially inwards by ∼11 å in the virion (red) relative to the assembly intermediates. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 6 Fit of the G protein pentamer into cryo-EM density. The procapsid density is shown in (a) and (b) with the truncated G pentamer rotated by 20° relative to its orientation in the virion. The unrotated G pentamer is shown fitted into the provirion EM density in (c) and (d). Electron densities are color coded as described for Figure 5b. The models show connected Cα-atoms in yellow. The views are from outside the virus looking down a five-fold axis (a,c) and with the five-fold axis vertical (b,d). Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 7 Fit of the F protein pentamer into cryo-EM density of the procapsid (a) and (b) and provirion (c) and (d), color coded as described for Figure 5b. The views are down a five-fold axis (a,c) and from the side (b,d). Only the truncated F protein is shown, that is the β-barrel without the EF and HI insertions. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 8 Cryo-EM density (blue) of the procapsid with an icosahedral two-fold axis vertical. The difference map of the procapsid minus the provirion cryo-EM density (in red), shows the presence of the B scaffolding protein on the two-fold axis. G pentamer and F pentamer structures are shown on either side of the two-fold axis. Note the additional density outside the F capsid proteins, which has been assigned to the D scaffolding proteins. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 9 An asymmetric unit (red) has been superimposed on the surface density of the procapsid after subtracting all but the putative D protein density. The position of the D tetramer (D1D2)2 is indicated in blue outline, illustrating the differing environments for the D1 and D2 monomers. The D tetramers form a cohesive scaffolding by themselves that serves to stabilize the procapsid prior to DNA insertion. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)

Figure 10 Diagrammatic representation of the protein organization in the procapsid, provirion and virion. Structure 1995 3, 353-363DOI: (10.1016/S0969-2126(01)00167-8)