Fig 1.
(A) Schematic diagram of the late operon of bacteriophage 80α. ORFs (arrows) corresponding to tail and baseplate proteins are colored tan (MTP), green (TMP), red (Dit), pink (Tal), purple (RBP), light blue (FibL), gray (Hyd), and dark blue (FibU). TerS, small terminase; TerL, large terminase; PP, portal protein; gp44, ejection protein; SP, scaffolding protein; CP, capsid protein; Hol, holin; Lys, lysin. (B) Schematic diagram of the baseplate, colored as in A. The question mark indicates an observed density of uncertain identity, probably corresponding to the Tal CTD. (C) Cryo-electron micrograph of 80α tails collected on a Titan Krios microscope with a DE-20 detector. Scale bar = 50 nm. (D, E) 2D class averages, showing the baseplate in side (D) and top (E) views, viewed parallel and perpendicular to the long axis of the tail, respectively. A thin fiber is indicated with a yellow arrow in E. (F) Fourier shell correlation (FSC) curve between two half datasets for the C6 (blue) and C3 (red) reconstructions. The resolution at FSC = 0.143 (gray line) is indicated for each.
Fig 2.
Reconstruction and modeling of the 80α baseplate.
(A) Isosurface representation of the 80α baseplate C6 reconstruction at density cutoff level of 4.5 standard deviations above the mean (4.5 σ), viewed from the side and bottom, segmented and color-coded according to the scheme in Fig 1. (B) Cutaway side view, showing the inside of the tail. Peripheral structures are shown as a transparent surface. (C) Side view at lower density cutoff level (3 σ), showing the tail fibers, tail rings and rod density more clearly. (D) Ribbon representation of the atomic model of the entire baseplate, viewed form the side and bottom and colored as above. (E) Model with all except one peripheral structure removed to show MTPU, MTPL, Dit and Tal underneath. (F) Complete baseplate model showing the full-length FibL pseudo-atomic model.
Table 1.
List of 80α tail and baseplate proteins, showing ORF numbers in 80α and corresponding numbers in ϕ11, protein name, number of amino acid residues and calculated molecular weight, number of copies per particle, and a description of protein functions and location in the virion.
Table 2.
HHpred analysis of 80α baseplate protein sequences.
The most relevant hits are shown for each protein, with matched residues, PDB ID and chain identifier, HHpred probability (%), E-value and percent sequence identity in matched region.
Fig 3.
(A) One asymmetric unit of the sixfold symmetric baseplate core, showing MTPU (brown), MTPL (tan) and Dit (red). The stacking loops of MTP and Dit, the C-arm of MTPL and the tail binding loop of Dit are indicated. (B) Cutaway electrostatic surface showing the charge distribution inside the tail, colored from red (negative) to blue (positive). The cut surfaces of MTP and Dit are colored light and dark gray, respectively. The surface is colored from –10 (red) to +10 (blue) kcal/(mol*e) according to the color bar. (C) Superposition of MTPU onto MTPL (gray). MTPL (tan) shifted by the same amount then superimposes on Dit (red). The expanded view shows the superposition of the Dit tail binding loop (red) with the MTP C-arm (tan). The side chains of the triplet of residues conserved between the C-arm of MTP (Y159,R160,F161) and the tail binding loop in Dit (Y100,R101,F102) are shown in stick representation and labeled. (D) Detail of the interaction between Dit (red) and the RBP coiled-coil stem region (purple), showing the RBP binding loop in the Dit CTD. (E) Same view as D with RBP shown as a van der Waals surface colored from most hydrophilic (tan) to most hydrophobic (purple), according to the Kyte-Doolittle scale. The rotated view shows the N-terminal end of the RBP trimer and its interaction with the RBP binding loop, with side chains shown in stick representation. (F) Surface representation of Dit (red) and Tal (pink), viewed from the side. (G) Surface representations of Dit (top) and Tal (bottom) rotated 90° in opposite directions to show the interacting surfaces. One subunit each of Dit (red), Tal (pink) and TMP (green) is shown in solid color, the rest are colored by hydrophobicity as in E.
Fig 4.
(A) Isosurface representation of the C3 symmetric baseplate reconstruction at 4.5 σ density cutoff. The density corresponding to the Tal trimer is shown in pink, with the rest of the reconstruction in transparent gray. (B) Central section through C3 reconstruction (gray scale). Density inside the lumen presumably corresponding to TMP is indicated with the yellow arrow. Scale bar = 100Å. (C) The Tal density alone, viewed from the side and bottom. The disordered density at the bottom in the side view is assumed to be the Tal CTD and was removed from the bottom view for clarity. (D) Ribbon representation of the atomic model of Tal viewed from the side and bottom. One subunit is colored dark gray. The TMP model is shown in green. (E) Tal subunit, colored in rainbow colors from blue (N-terminus) to red (C-terminus). Relevant structural elements are labeled. (F) Density corresponding to the central part of Tal with the atomic models of Tal and TMP shown in pink and green, respectively. (G) View down the axis of the baseplate showing the inside of Tal as a van der Waals surface, colored from hydrophilic (tan) to hydrophobic (purple). TMP is shown in green. (H) Detail of the interaction between the TMP C-terminus and Tal. The three C-terminal residues of TMP (Y1152, Y1153 and L1154) are shown in stick representation.
Fig 5.
(A) Atomic model of the 80α RBP homotrimer. The three subunits are colored purple, blue and green. The stem, hinge, platform and two tower domains are indicated, as is an Fe atom (orange) coordinated by six His residues in the stem domain. (B) Superposition of 80α (purple) and ϕ11 (gray) RBPs, aligned by the tower and platform domains and rotated to emphasize the differing orientations of the stem domains. (C) Superposition of 80α RBP (purple) with the tail fiber (gp17) of P68 (gray), aligned by the tower domains. (D) Ribbon representation showing FibL (light blue), FibU (dark blue) and part of the RBP stem (gray). The positions of the K169 residues in the three FibL subunits are shown as red balls to indicate the relative shift of the three α-helices in the coiled coil. The C-terminal residue modeled in each FibL subunit is numbered. (E) Rotated view of D with the three FibL subunits colored green (p1), pale blue (p2) and blue (d). The two FibU subunits are in dark blue and magenta. RBP is gray. (F) Top view of baseplate showing the FibL ring octadecamer (colored as in E) and the MTP hexamer underneath (tan). (G) Isosurface (cutoff 3.0 σ) of a reconstruction made from signal-subtracted images excluding density corresponding to MTP, Dit, Tal, RBP and FibU. FibL density is blue. Additional density in the center (gray) probably corresponds to TMP. (H) Model for the complete FibL protein trimer built into the density from G. The lower panel is colored as in E, with structural elements labeled (CC, coiled coil; ϕK is the phage K gp68-like domain). (I) Top view of the dodecameric FibU ring, colored as in E.
Fig 6.
Model for conformational changes in the baseplate during the infection process.
(A) Initial binding of RBP to WTA. FibL and FibU may also be involved in binding to other, as yet unidentified surface structures (?). (B) Conformational changes in RBP lead to exposure of enzymatic activities associated with Hyd and Tal, allowing degradation of the cell wall peptidoglycan (PG). (C) Penetration of the plasma membrane (PM) by the Tal rod helices triggers release of TMP, leading to ejection of DNA through the central channel.