Extended Data Fig. 5: AlphaFold-guided design of modified tail fibres for use in retargeting PVCs.

a, Domain organization of the PVC tail fibre gene (pvc13). The tail fibre contains an N-terminal domain (NTD) with homology to tail fibres from other CISs, a domain that maps to shaft domains of adenoviral fibres, and a C-terminal domain with homology to the host-binding domains of bacteriophage tail fibres. Domains were depicted based on statistically-significant HHpred hits (>95%) and are drawn roughly to scale. Scale bar, 100 aa. b, Pvc13 is loaded via the NTD, implicating the C-terminal phage fibre tip domain as the cell-binding domain. Pvc13 was truncated at either end and loading was determined via denaturing western blot on purified PVC particles. Only truncation of the NTD resulted in a loss of Pvc13, suggesting this domain connects the tail fibre to the PVC. An additional blot against the payload (Pdp1-NTD–Cre) was included to confirm the presence of assembled PVCs. c, The C-terminal phage fibre tip domain of Pvc13 shares structural and sequence similarity with known receptor-binding domains from bacteriophages. Structural superpositions were generated between the phage fibre tip domain from Pvc13 (in grey) and analogous regions from a prophage tail fibre in Bizionia argentinensis (cyan; PDB: 6OV6), gp10 from phage T4 (magenta; PDB: 5IV5) and gp12 from phage T4 (yellow; PDB: 5HX2). d, Predicted structures and delivery characteristics of wild-type and engineered PVC tail fibres. The AlphaFold predicted structure of the C-terminal phage fibre tip domain contains a helical tube structure with a globular tip on one end that we hypothesized to be the distal target recognition domain of the overall tail fibre. Importantly, there also exists a short 32 aa region (labelled CTD; depicted in gold) that loops back through the tube, likely stabilizing it. We observed that designs lacking this CTD (when co-purified with the PVC complex) produced misleading activity in Cre delivery experiments in HEK 293FT, perhaps as a result of sporadic ejection of the Cre payload from the PVC particle (free Cre protein can enter cells independently of a delivery vehicle⁵⁰). However, designs retaining this CTD produced no aberrant signal in HEK 293FT, indicating payload ejection was once again properly regulated. Amino acid sequences for representative Pvc13 designs can be found in Supplementary Tables 1–4. Scale bar, 150 μm.