## 4 Structural Features of the Conserved _exo-xis_ Region Proteins
### The Orf60a Gene of \(\lambda\) and \(\text{Stx}^{+}\) Phages  
After infection by phage bearing a deletion in _orf60a_, there are more bacterial survivors and more lysogens among them suggesting that _orf60a_ serves a pro-lytic role during development. In support of this role, the induction of lysogens after activation of the SOS response by antibiotics or oxidative stress takes longer to proceed in _orf60a_ mutants. All of these effects are more pronounced for \(\Phi 24_{\text{B}}\)\(\Delta\)_orf60a_ mutants than \(\lambda\)\(\Delta\)_orf60a_ mutants.  
The AlphaFold and RoseTTAFold predictions for Orf60a superimpose well with a \(\mathcal{C}\alpha\) backbone RMSD of 1.84 A. Each model is described by three or four anti-parallel \(\beta\)-strands serving as a platform for one \(\alpha\)-helix. Compared to the AlphaFold model, the helix in the RoseTTAFold model is shorter and does not extend past the \(\beta\)-sheet. Since a helix with no contact is likely to be unstable in the absence of a ligand, the RoseTTAFold model was chosen for further analysis.  
When the predicted structure of Orf60a was compared to the protein structure database, it compared favorably to a family of small DNA binding domains (DBDs) that use a \(\beta\)-sheet to interact with the major groove [47; 48; 49; 50]. This family included the GCC box DNA binding domain from _Arabidopsis_ AtERF (PDB:1 GCC, RMSD = 1.97 A; 8% identity), the bacterial cell division reactivation factor CedA (PDB:2 BNS, RMSD = 2.16 A, 23% identity), the \(\lambda\) phage amino-terminal integrase DBD (PDB:1 KJK, RMSD = 1.67 A, 11% identity), and the Tn916 transposon DNA binding domain (PDB:1B69, RMSD = 1.76 A, 3% identity).  
While the true structure, function and mechanism of Orf60a is unknown, its potential role in gene expression aligns with its ability to serve the lytic program, especially in \(\text{Stx}^{+}\) phages.  
Even though there was practically no sequence identity between the Tn916 structure and the \(\lambda\) Orf60a model, it was selected for a comparison because Tn916 was solved in its DNA-bound state. This comparison is presented as a sequence and secondary structure alignment in Figure 4a and as a refined \(\lambda\) Orf60/DNA model in Figure 4b. The \(\lambda\) Orf60a putative DNA binding domain uses all four \(\beta\)-strands to make DNA contacts with a mixture of basic amino acids at the periphery and polar/aliphatic amino acids within the major groove. While the complement of Tn916 contacts to DNA are different in their location within the sequence, they loosely follow the same pattern of basic amino acids at the periphery and aliphatic/aromatic amino acids within the groove.  
Figure 3: Molecular modeling the conserved _exo-xis_ region proteins in their monomeric forms from bacteriophages \(\lambda\) and \(\Phi\)24\({}_{\rm B}\). (**a**) Sequence alignments are shown for Orf60a, Orf63, Orf61, and Orf73. Identical amino acids shaded grey. There are two paralogs of Orf63 in \(\Phi\)24\({}_{\rm B}\). Only the C-terminal domain of Ea22 is shown. The predicted secondary structure is presented below the sequences with helices in red and strands in blue. (**b**) Molecular models of selected _exo-xis_ proteins by AlphaFold. (**c**) Molecular models of selected _exo-xis_ proteins by RoseTTAFold. Structural similarity (as backbone RMSD) and accuracies (as pLDDT values) between the AlphaFold and RoseTTAFold models are indicated. Due to the structural dissimilarity, residues 1-23 are not included in the statistics of the \(\Phi\)24\({}_{\rm B}\) Orf61 model.