Figure 1.
Cryo-ET structures of individual wt PRD1 with a tail tube.
(A, Left) PRD1 capsid organization represented as in [8] with each of the 4 P3 MCPs trimers composing the asymmetric unit coloured in green, cyan, blue, and yellow; in red is the vertices penton protein P31 (PDB ID 1W8X). The white triangle marks a virus facet, and 2, 3, and 5, respectively, the 2-, 3-, and 5-fold icosahedral symmetry axes. (A, Right) Schematic presentation of wt PRD1 architecture according to the current knowledge (numbers in parentheses identify the corresponding protein structures in the Protein Data Bank). (B) Overall view of a section of a reconstructed tomogram with the different wt PRD1 related structures (DF, DNA-filled particle; OT, orthogonal tube; LT, longitudinal tube); some semi-empty particles display a darker region within the membrane indicating residual DNA (DNAr). (C) Cryo-image of PRD1 with a tube at 50,000× magnification (2 µm under focus) showing the bilayer structure (inset) of the vesicle (white arrowheads) and of the tube wall (gold arrowheads). (D, Top Left) Tomographic central xy section of a representative wt PRD1 particle with a tube possibly at initial stage of DNA ejection; red arrowheads indicate the vertex aperture from where the tube protrudes and the black arrow the conical tip of the tube. A semicircular red line within the vesicle marks the presence of residual genome. (D, Top Right) An orthogonal view (xz) of the tube. (D, Bottom Left and Right) As corresponding top but with a wt PRD1 particle with a tube possibly at the final stages of the DNA ejection with the vesicle showing a “map pin” morphology. (E) One individual tube with the long axis (quasi-)orthogonal to the tilting plane. (E, Left) Density pattern of the tube from a central tomographic section (xy) and (E, Right) xz section of the tube. White double head-arrows mark the dimensions of the different particle structural elements; tomograms were denoised using TOMOAND [58] and displayed in AMIRA (Visage Imaging GmbH, Berlin).
Figure 2.
Apertures at the 5-fold vertices in wt PRD1 capsid.
(A, Left) Central tomographic section of a wt PRD1 particle with a tube showing two contiguous de-capped vertices (red arrowheads) and nearby the shrinking of the vesicle volume within the capsid (denoised as in Figure 1D). (A, Right) Central section of the subtomogram averaged wt PRD1 capsid displaying one of the de-capped vertices (red arrowhead); not to scale with previous. (B, Left) Isosurface representation of the nonicosahedrally symmetrised subtomogram averaged PRD1 with tube volume (capsid blue, membrane/tube gold; displayed at 1.5σ in Chimera [59]) with the atomic models of the vertex complexes, the peripentonal P3 MCPs trimers (yellow), the P31 penton protein (red), and the membrane protein P16 with its transmembrane helices (magenta) (PDB code 1W8X) at the three contiguous de-capped vertices joined by white lines (vertex-to-vertex ∼35 nm). (B, Right) Cut-through of the PRD1 particle isosurface (rotate by 180° from left) showing the missing density of the vesicle regions proximal to the de-capped vertices (contoured at 0.35σ). Hereafter, the boundaries between capsid shell, vesicle, and tube were assigned by eye and prior knowledge [8]; the structural analysis of the PRD1 atomic model in the context of our subtomogram averaged maps was carried out as described in Material and Methods. (C, Left) Enlarged view of one of the de-capped vertices (contoured at 1.1σ) whose aperture matches the loss of the spike complex; (C, Right) As left but subtomogram averaged PRD1 tube density with 60-fold symmetry imposed (pale-green, contoured at 2.5σ). (D, Left) Cut-through of the nonicosahedrally symmetrised PRD1 tube density (displayed at 0.5σ; capsid in blue, vesicle and tube in gold) superimposed onto the PRD1 reference model (semitransparent light-grey density contoured at 1.2σ with labelled atomic models of P3, P31, and P16 coloured as in B) showing the size agreement of the corresponding capsid shells but not of the vesicle. (D, Inset) Enlarged views showing the spatial relationship between the peripentonal P3 MCPs trimers, P31 pentons, and P16 proteins relative to the vesicle densities, respectively, of PRD1 icosahedral cryo-EM map (Top), of 60-fold symmetrised averaged cryo-ET map (semitransparent pale-green) (centre) and of nonicosahedrally symmetrised PRD1 tube density (bottom); in the latter two cases it is apparent the loss of P16 membrane interactions with the vesicle.
Figure 3.
Vesicle morphology in PRD1 procapsid.
(A, Left) Gallery of 2D cryo-images of vesicle shapes (top, stomatocyte-like; bottom, discocyte-like) within the PRD1 procapsids; scale bar, 20 nm. (A, Right) A cartoon of the steps leading to vesicle transformation: (1) capsid is intact and vesicle not exposed to environment, (2) opening of one or more vertex complex (by in vitro or in vivo triggering) destabilizes the vertex–vesicle interactions and directly exposes the vesicle to surrounding conditions, and this induces the shape transitions (3 and 4). (B) Gallery of 2D cryo-images of PRD1 procapsids (scaled to A, Left) with a tube with characteristic views. (B, Top) Particles with forming tube viewed along the long axis of the tube with insets highlighting two possible tubulation stages: (i) scission (red arrows indicate the contact points to the vesicle) and (ii) tube release; both stages are schematically represented by the cartoon on the left. (B, Centre) Particles with a tube viewed orthogonal to the long axis of the tube. (B, Bottom) Additional views of particles with a tube. (C) xy, yz, and xz sections of two different PRD1 procapsids cryo-ET reconstructions (side-by-side) unequivocally showing that the budding of the tube occurs within the capsid and tangentially to the viral vesicle and recapitulating the views of 2D cryo-images in (B). (D, Top) Isosurface representation of the 3D volume of the procapsid shown in (C, Right) with docked the 12 vertex complexes (as Figure 2B and 2C) viewed along the icosahedral 2-fold axis and 90° rotated (D, Bottom). (E) 2D images of procapsids with a membrane with two budding tubes viewed along their long axis (scaled to A, Left).
Figure 4.
Subtomogram averaging of the PRD1 DNA exit gate.
(A, Left) Cut-through of the isosurface of the subtomogram averaged cryo-ET volume of the PRD1 DNA exit gate (semitransparent, white-smoke; clipped surface in dark-grey contoured at 1.1σ) with superimposed MCP P3 trimers (represented in cartoon and color-coded as in Figure 1A, left) next to the de-capped vertex and with a space-filled model of a B-DNA (red and blue) shown within the cavity of the tube. (A, Bottom Left) The corresponding central section (0.88 nm thickness) with the red arrowheads pointing at the density at the interspace between the vesicle and the capsid, which is linearly connecting the vesicle and the tube (scale bar, 5 nm). (A, Right) Cut-through view of the density corresponding to the external tube stemming from the de-capped vertex (darker grey area). (B) Superimposition of the exit gate reconstruction (white smoke and dark-grey) onto the PRD1 cryo-EM density (semitransparent light-grey; contoured at 0.9σ) with hexameric models of the first viral ATPase of a nontailed virus (represented as cartoon in magenta and orange) [36] manually fitted in two putative loci along the tube density indicating the mismatch in size between the diameters of tube and the putative hexameric ATPase as highlighted in the inset.
Figure 5.
Rotational analysis of the tube and tube morphology.
(A) Examples of a cryo-image of wt PRD1 (∼6 µm under focus) (Left) and a negative stain image of PRD1 procapsid (∼4 µm under focus) (Right) with orthogonal views of the tube. The insets (4× magnification) highlight the density pattern of the orthogonal 2D projection of the tube. The density of the phage across the figure has been inverted (white) for clarity. Scale bar, 10 nm. (B, Top Left) Plot of the averaged rotational spectra corresponding to the subpopulations for which the 3-fold harmonic component was dominant with correspondent variance (Top Right) and resulting 2D averaged density pattern (Bottom Right) obtained by the KerdenSom analysis of 1,758 rotational spectra of 2D cryo-images of orthogonal tubes boxed out from PRD1 procapsids. (B, Bottom, Left) A pie-diagram summarising the distribution of rotational symmetries (see also Figure S4). (C, Top Left) Consecutive representative z-slices from left to right starting at slice 20 (0.88 nm thickness) of the tube volume obtained by averaging 33 subtomograms (average class 2) (white, density pattern). (C, Top Right) As left but averaged tube obtained from 64 subtomograms (average class 3). (C, Insets) Central cross-sectional density pattern with high- and low-contrast regions marked respectively by a red dot and a cyan arrow. (C, Bottom) Central yz section corresponding to the tube average maps with gold and dark-gold arrowheads pointing at the ring-like structure (see also Figures 7 and S5). Both averaged volumes were low-pass band filtered at 5.0 nm before displaying. (D) Mean density profiles along z (Top) calculated from the central xz section of the class 2 (gold line) and class 3 (dark-gold line) averaged tubes (Bottom). Dimensions of the tube parameterized as hollow cylinder (r1, inner radius; r2, outer radius; r, average radius) have been given for the averaged tube 2.
Figure 6.
(A) Tomographic slices of an Escherichia coli cell infected by wt PRD1 (∼30 min p.i.). Scale bar, 50 nm. (A, Left) Full particle, outlined in a black line, with forming tube crossing the outer membrane (OM). Inner plasma membrane (IM) and peptidoglycan layer (PL) are indicated. (A, Inset, Top Left) Particle viewed through three intersecting planes marked in green, blue, and magenta with corresponding blue and magenta planes on the right. (A, Inset, Below Left) A segmentation of the particle colour coded as Figure 2B, left with superimposed density derived from the atomic model (PDB code 1W8X) in light-blue, in red the outer and inner membranes, and cyan the peptidoglycan layer. (A, Centre) Semi-full particle, outlined in a black line. (A, Centre, Inset) A tomographic slice (Left) with superimposed segmentation of the capsid and cell envelope (Right). The white arrowhead indicates additional openings of the capsid. (A, Right) An almost empty particle, outlined in a black line. (A, Right, Inset) Tomographic slice showing a particle with a “map-pin” shape vesicle and full-length tube penetrating the cell envelope (Left) and corresponding segmentation (Right). (B) Tomographic slices of epon sections showing a DNA-filled wt PRD1 particle with tube far away from cell (Left; 5 min p.i.) and a DNA-devoid wt PRD1 with the tube closer to the cell (Right; 30 min p.i.) both with DNA visible as a darker linear density within the tube as marked by the white arrows; scale bar, 20 nm. (C) Sequential tomographic z-slices (top left corner) of a wt PRD1 particle (epon embedding; slice thickness 0.44 nm; 30 min p.i.) proximal to the outer membrane and inducing membrane deformation (see also Movie S2). Scale bar, 20 nm. Red lines in the insets below outline the drastic changes in cellular membrane morphology as seen above.
Figure 7.
Schematics of PRD1 genome translocation via tunnelling nanotube and model of PRD1 membranous tail tube structure.
(A) Sequential steps of PRD1 infection and tube formation. Step 1, PRD1 particles approach the cell surface in a random orientation. Step 2, after binding to the cellular receptor, partial rolling/tumbling of the particle due to solvent movement and/or binding of further viral protein P2 or secondary binders such as P5 proteins to cellular receptors guide the re-orientation of the unique vertex almost orthogonal to the cell. De-capping of the cell-interacting vertex triggers the membrane reshaping as a consequence of the loss of vertex-stabilizing protein P16 interactions and the influx of components of the extracellular milieu through the opened vertex (cf., change in osmolarity). Step 3, the vesicle transformation leads to the tube polymerization and piercing of the cell envelope as well as digestion of the peptidoglycan layer by virion-associated enzymes [30]. Once in the cytoplasm, the tip of the tube unplugs and the DNA is translocated relative to the tube from the vesicle into the cytoplasm. (A, Inset) Matching of the ring-like structure (black arrowheads) within the capsid region of the gate volume (white-smoke). (B, Left) Isosurface representation of the averaged model tube 2 (n = 33, Figure 5C, Left) with the distinctive ring-like structure nearby the top end of tube (low-pass band filtered to 5.0 nm and contoured at 0.8σ; gold) with a model of B-DNA fragment manually docked within the channel viewed perpendicular to the tube long axis. (B, Centre) As left but displayed at higher contouring level (1.4σ) (Top) and 90° rotated (Bottom). (B, Right) As centre but viewed through a cutting-plane halfway into the tube (Top) and with a 90° rotated view of the tube cut-through above the tip-end (Bottom). For a superimposition of the averaged density of tube 2 onto average tube 3, see Figure S6. (C) Putative tube's architecture. (C, Top) Stereo-view of a portion of the averaged tube 2 density (as mesh contoured at 1.4σ; 90° rotated relative to B, Top) cut-through its longitudinal axis with manually fitted a lipid bilayer model (cyan) and a membrane protein (blue). (C, Bottom) Stereo-view of the architecture of the tube as depicted by filling the averaged density of tube 2 with a net of atoms (gold) representing the membrane bilayer in turn decorated by scaffolding membrane proteins (blue). These proteo-lipid upright strands are connected by weaker lateral contacts that confer to the highly curved tube wall the fenestrated appearance.