Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1. Location of the mab 6F10 epitope in capsids.

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1 Supplementary Figure 1 Location of the mab 6F10 epitope in capsids. The epitope of monoclonal antibody, mab 6F10, was mapped to residues A862 H880 of the HSV-1 major capsid protein VP5 by immunoblotting experiments, and the capsid-antibody complex visualized by cryoem of 2M GuHCl-treated B-capsids, called Gcapsids (Newcomb, W.W. & Brown, J.C., J Virol. 65, , 1991), that had been incubated with antibody (Spencer, J.V. et al., Virology 228, , 1997). G-capsids have lost pentons and the hexon-capping VP26 molecules as well as peripentonal triplexes and internal scaffolding domains (Booy, F.P. et al., Proc Natl Acad Sci U S A., 91, , 1994). Consequently the VP5 epitope is present only in the uncapped hexons, and mab density was observed near the tips of these hexons, just inside the openings of the transcapsomeric channels (Spencer, J.V. et al., Virology 228, , 1997). The fit of the VP5 upper domain fragment (yellow ribbons structure) is shown for (a) the penton, and (b) a hexon of the HSV-1 capsid map, with the mab 6F10 epitope colored in red. The position of the epitope in our fit, on the exterior rim of the trans-axial capsomer channel that runs through to the interior of the capsid, is consistent with the localization by antibody, providing conclusive validation of our cryoem density maps. We also predict that pentons with epitopes exposed may bind mab 6F10 more avidly than hexons where access to the epitope is partially blocked by the VP26 molecule (colored green).

2 Supplementary Figure 2 Estimating resolution of cryo-em density maps. (a) Fourier Shell Correlation (van Heel, M., Ultramicroscopy 48, , 1987) plots drop below 0.3 at 6.8 Å resolution for the HSV-1 map, and 7.1 Å for the PRV map we use this level as a compromise between the overly-conservative 0.5, and the gold-standard value of (Scheres, S.H. & Chen, S., Nat Methods 9, 853-4, 2012; Henderson, R. et al., Structure 20, , 2012). (b) The VP5 upper domain crystal structure (PDB 1NO7 Bowman, B.R., et al, EMBO J. 22, , 2003) in ribbons form colored as a rainbow from E484 (blue) to T1054 (red), and in yellow as fit into the HSV-1 cryoem density (blue translucent surface). (c) Surface renditions of the atomic model (PDB 1NO7) with different resolution limits applied, as indicated, using the molmap function of UCSF Chimera (Pettersen, E.F. et al., J Comput Chem. 25, , 2004) based on the pdb2mrc program in the EMAN package (Tang, G. et al., J Struct Biol. 157, 38-46, 2007). Comparison of various features with the cryoem surface in (b) supports the resolution estimated in (a).

3 Supplementary Figure 3 Organization of the major capsid protein VP5. (a) Secondary structure prediction with PSIPRED (Jones, D.T, J Mol Biol. 292, , 1999) together with the constraint of the upper domain crystal fragment location suggests an organization of the VP5 protein as depicted, with the lower and middle domains at either end, bracketing the upper domain. Elements of the HK97-like fold that are consistent with the lower domain correspond well with the secondary structure predicted for the VP5 N-terminal, excepting an insertion (extra density 1: ~ ) and the transition through the middle domain (~ ) to the onset of the upper domain fragment. (b) The N-terminal-most 50 amino acids are proposed to form the sub-penton tubes visualized in the HSV density map (red) and the curved tubes beneath hexons (see Fig. 4 in the main text). The PSIPRED prediction for this region includes a 20-residue helix. (c) Several of the density elements are superimposed on our VP5 lower domain model (see Fig. 3b in the main text) including regions corresponding to the phage HK97 capsid protein model. (d) The C- terminus of the upper domain fragment (PDB 1NO7 Bowman, B.R., et al, EMBO J. 22, , 2003) leads into a region predicted to be -rich, corresponding with a feature of the HSV-1 density map as indicated. The remaining middle domain density is difficult to interpret in terms of connectivity and secondary structure elements, consistent with the paucity of helix predicted for the remainder of VP5 until the C-terminal-most ~50 residues.

4 Supplementary Figure 4 Secondary-structure prediction for pul25, pul17, and pul36 sequences from HSV-1. Amino acid sequences were analyzed by PSIPRED (Jones, D.T, J Mol Biol. 292, , 1999) using the server at the UCL Department of Computer Science (Buchan, D.W., et al, Nucleic Acids Res. 41, W349-57, 2013). Purple shading indicates prediction for -helix, while yellow is for -strands. (a) pul25, where the N-terminal 133 residues not included in the crystal structure (Bowman, B.R. et al., J Virol. 80, , 2006) reveal the likelihood of adopting a 60-residue helix (H1), consistent with the length of the CVSC helical bundle. (b) pul17 includes several regions likely to be helical and possibly contributing two parts to the CVSC bundle domain. Since the motif is likely to be a helix-turn-helix, with no significant domain at the turn, we favor the helices indicated (H2 and H3). (c) pul36 C- terminal region has one candidate (H4) for interacting with the CVSC bundle at the C-terminal-most part. Note that any other contribution to the bundle would require two helices (there and back). However, we caution that no -helix or structure is predicted for a sizeable region of the C-terminal fragment, and we note 14 tandem repeats of the PQ pair within this region. Both features are unusual, and interpretation of the sequence and/or the structure prediction may need revisiting in light of any revisions.

5 Supplementary Figure 5 Comparison of pul25 binding on the PRV and KSHV capsids. (a) Fits of the HSV-1 pul25 crystal structure (PDB 2F5U Bowman, B.R. et al., J Virol. 80, , 2006) are shown around the penton (blue) for PRV, at left, and at right for Kaposi Sarcoma herpesvirus (KSHV, EMD 6038 Dai, X. et al., Proc Natl Acad Sci U S A 112, E649-56, 2015). The top row shows the context of the cryoem-derived density maps, including disposition of the CVSC helical bundles in orange (PRV) and yellow (KSHV), while the bottom row shows the pul25 model (ribbons) around schematically-represented capsomers. Note that the orientation of the penton subunit fits (PDB 1NO7 Bowman, B.R., et al, EMBO J. 22, , 2003) reveals no significant change in orientation, whereas the pul25 subunit fits are considerably displaced. (b) Superposition of the pul25 fits into PRV (red) and KSHV (green) density reveals the magnitude of the displacement to be ~30. Note that in PRV, pul25 approaches 2 copies of the penton VP5 subunit, while the KSHV analog, porf19, approaches only one copy. (c) Comparison of the fits reveals that the orientation of the pul25 analogs are ~180 rotated relative to the N-terminal domain. Contacts between the KSHV protein and the capsid are therefore completely different than in PRV or HSV, despite the fold being common.