Structural differences between CP-A and CP-B in GLV-HP and GLV-CAT.

The overall RMSD values were calculated to evaluate the structural similarity. The major core domain are well aligned with RMSD = 0.401 Å across 773 Cα pairs between CP-As of GLV-HP and GLV-CAT, and 0.396 Å across 835 Cα pairs between CP-Bs of GLV-HP and GLV-CAT. Conformational changes were observed in the regions of C-terminal extension in CP-A and variable loop in CP-B, as indicated by yellow dotted circles. (TIF)</p

Representative raw micrographs of GLV-HP and GLV-CAT particles.

Representative raw micrographs of GLV-HP and GLV-CAT particles.</p

Lack of putative cap-snatching pockets in GLV and OmRV.

(A) Electrostatic surfaces of Totiviridae and toti-like virus CPs are shown in blue (positive) and red (negative) scales. The CP of the yeast Totiviridae viruses ScV-L-A and ScV-L-BC have cap-snatching active pockets and invariant His residues (His154 or His156) (yellow dotted circles). The CP of TVV2 is speculated to have a cap-snatching pocket with three putative His residues (His537, His648, and His658) in a position similar to that in the yeast Totiviridae viruses (yellow dotted circle). However, in the GLV and OmRV CPs, no His residue is observed on the corresponding cap-snatching pocket, although three His residues (His288, His387, His444) in the GLV CP and one His residue (His986) in the OmRV CP are exhibited on the surfaces. (B) Conserved helices and invariant His residue in Totiviridiae and toti-like viruses. A conserved helix-turn-helix and a long helix are highlighted as dotted circles and rectangles in each CP structure.</p

Pore structure of the GLV capsid.

A surface electrostatic potential map of the 5-fold CP complex of GLV-HP and GLV-CAT from outside and inside views is shown in red (negatively charged) and blue (positively charged) scales. A close-up view and cross-section of the pore structure and surface charges are also shown at the bottom.</p

Atomic model of capsid and CPs in GLV-HP and GLV-CAT.

The T = 1 capsid geometry and CP-A and CP-B organization in (A) GLV-HP and (H) GLV-CAT. Raw cryo-EM micrographs of (B) GLV-HP and (I) GLV-CAT. The close-up views of N-termini and C-termini in CP-A and CP-B are shown in (C–G) for GLV-HP and (J–N) for GLV-CAT.</p

C-terminal extension structure of the CP–CP interface in the GLV-HP and GLV-CAT capsids.

The atomic model of the 5-fold CP complex of (A) GLV-HP (CP-A in green; CP-B in orange) and (C) GLV-CAT (CP-A in light blue; CP-B in pink) is shown from their inside view. The interlocking C-terminal extensions from CP-A to CP-B and those from CP-B to CP-A are highlighted in the surface representation. The close-up views of the interactions between the C-terminal extension and the adjacent capsid are shown in (B) for GLV-HP and in (D) and (E) for GLV-CAT. The amino acid residues involved in the interactions are indicated. The red dashed lines between the amino acid residues indicate predicted hydrogen bonds with their distances in Ångström.</p

Assigned amino acid residues and the secondary structure diagram of the atomic model of CP-B of GLV-HP and GLV-CAT.

The rainbow color begins with blue at the N-terminus (Pro1) to red at the C-terminus (Val929). Considering the predicted internal IRES sequence in the GLV genome, the translation of ORF1 (CP) does not initiate from the first methionine but from an internal amino acid residue. The first amino acid residue of the CP was started from the internal Pro residue (PENIT …), according to a previous mass spectrometry analysis of the purified GLV particles. (TIF)</p

Overall and local resolution estimations of GLV-HP and GLV-CAT reconstructions.

(A) FSC curves and (B) local resolution of the final cryo-EM 3D reconstruction for GLV-HP and GLV-CAT. (TIF)</p

Structural comparison of CP-A and CP-B.

The CP-A and CP-B of GLV-HP are colored in green and orange, and those of GLV-CAT are colored in light blue and purple. The total RMSD values between CP-A and CP-B were calculated for GLV-HP and GLV-CAT. Some conformational changes are observed in the regions indicated by red dotted circles. The major core domain apart from N- and C-termini, and red dotted circled regions, are well aligned with RMSD = 0.618 Å over 684 Cα pairs between GLV-HP CP-A and CP-B), and 0.618 Å over 732 Cα pairs between GLV-CAT CP-A and CP-B while other loops and interfaces contribute to the overall higher RMSD values (4.338 Å across all 775 Cα pairs in GLV-HP, and 8.863 Å across all 848 Cα pairs in GLV-CAT). (TIF)</p

Secondary structure-based amino acid sequence alignment.

Amino acid sequences of GLV-HP (PDB ID: 8R0F, chain A), OmRV-LZ (PDB ID: 7D0K, chain A), ScV-L-A (PDB ID: 1M1C, chain A), ScV-L-BC (PDB ID: 7QWX, chain A), and TVV2 (PDB ID: 7LWY, chain A) CPs were aligned using PROMALS3D multiple sequence and structure alignment server. Extra surface loops of CP in GLV (loops 1–5) and OmRV (loops 1–4) are boxed in the aligned sequences. (TIF)</p