Structure of the Ty3/Gypsy retrotransposon capsid and the evolution of retroviruses

Cryo-ET of PR- Ty3 particles. (A) Slices through the tomographic reconstructions of purified, plunge-frozen PR- Ty3 particles (shown as an average of 10 computation slices). A regular icosahedral T = 9 particle (Left) and an irregular particle with a variable T-number (Right) are shown. (Scale bars, 50 nm.) (B) Ty3 lattice maps visualized by placing hexagons or pentagons at the positions of capsomers. Note the uniform distribution of pentamers in the T = 9 particle (Left) and the uneven distribution of pentamers in the other particle (Right). Vectors connecting neighboring fivefold positions are shown as lines, and local T-numbers are indicated. Fivefold positions are colored blue, threefold positions are colored yellow, and pseudothreefold positions are colored green. (C) Subtomogram averages of the fivefold, threefold, and pseudothreefold positions within the Ty3 PR- particles. Within each structure, the fivefold position is colored blue, the threefold positions is colored yellow, and the pseudothreefold position is colored green. (D) Composite representation of complete Ty3 particles, colored radially from green (low radius) to blue (high radius).

Cryo-EM reconstruction of a T = 9 PR- Ty3 particle. (A, Left) Slice through the center of the PR- Ty3 particle reconstruction resolved at 7.5 Å. The outer layer with distinct α-helical densities is the CA layer, and the fainter blurred layer underneath likely represents spacer, NC, and the associated genome. (A, Right) Three-dimensional reconstruction of a PR- Ty3 particle colored radially from green (low radius) to blue (high radius). (Insets) Close-up views of the fivefold (yellow box), threefold (red box), and pseudothreefold (orange box) positions. Within the positions with different symmetries, the densities are colored to indicate the different domains of CA: Fivefold CA-NTD is colored green, CA-CTD is colored yellow, threefold and pseudothreefold CA-NTD is colored cyan/blue in conformation A/B, and CA-CTD is colored orange/red in conformation A/B. (B) Ty3 CA-NTD (cyan) and Ty3 CA-CTD (orange) homology models are shown superimposed on the HIV CA-NTD (purple; PDB ID code 4XFX) and HIV CA-CTD (pink; PDB ID code 4XFX) structures (Upper, in which helices are numbered) and the templates used for homology modeling: Arc N-terminal lobe (gray; PDB ID code 4X3I) and Arc C-terminal lobe (gray; PDB ID code 4X3X), respectively (Lower). (C) Rigid body fitting of the Ty3 homology models of the CA-NTD (cyan) and CA-CTD (orange) into the Ty3 cryo-EM map.

Ty3 CA-NTD and CA-CTD at higher resolution. (A, Left) Structure of a Ty3 CA-CTD dimer at 4.9 Å generated by alignment and averaging of nine non–symmetry-related copies of the Ty3 CA-CTD. The homology models of the Ty3 CA domains have been flexibly fitted into the refined EM maps. Protein models are colored from blue (N terminus) to red (C terminus). The bulky side chains of F134, R135, W138, R157, and Y164 amino acids in the CA-CTD are colored pink. (A, Right) Close-up view of the very C-terminal part of the CA-CTD from the complete Ty3 particle map, which creates one of the contacts between two neighboring CA monomers in the particle lattice. (B) Structure of a CA-NTD dimer at 5.5 Å generated by alignment and averaging of four non–symmetry-related copies of the Ty3 CA-NTD dimer, colored as in A. (C) As in B, but shown at a lower isosurface level to illustrate the unoccupied densities in the refined EM map of the CA-NTD dimer. These represent the very N-terminal parts of the Ty3 CA-NTD. The N-terminal part of the CA-NTD is shown as a string of beads in cyan (conformation A) and blue (conformation B). In both conformations, the N-terminal part of the protein first runs outward along the inner interface between the CA-NTD helix 1 and helix 2 (C, Right). In conformation B, it then continues over the top of the CA-NTD and down the outer side of the threefold position (C, Left).

Variability of Ty3 CA-NTD and CA-CTD relative orientations. (A) Model of a complete Ty3 PR- particle showing fitted protein models and EM density (transparent gray). Proteins are colored as in Fig. 3. (B) Close-up views of the different positions within the Ty3 particle. One monomer in a fivefold position is highlighted in black for clarity. (C) Superimposition of the nine independent copies of CA showing the large relative movements of the CA-NTD and CA-CTD about the flexible interdomain linker. The CA-CTD was used for alignment of the structures. (D) Superimposition of the nine independent copies of CA-NTD, together with their neighbors, showing three distinct relative orientations aligned on one CA-CTD (gray). (E) Superimposition of the nine independent copies of CA-CTD, together with their neighbors, aligned on one CA-CTD (gray), illustrating continuous variability of the dimer and trimer CA CTD–CTD interfaces.