The BASEL phage collection was developed to provide access to diverse bacteriophages, distinct from model phages. Escherichia phage JohannRWettstein (Bas63), a myophage in the collection, is a member of the subfamily Ounavirinae and the Felixounavirus genus. Using cryo–electron microscopy, we investigated Bas63’s structure to explore its evolutionary relationships and functional adaptations. Our structures reveal a series of gene products: (i) a capsid decorated with β-tulip proteins at three-fold symmetry axes and a Hoc-like protein at hexamer centers, (ii) a conserved connector with an additional 12-fold ring of collar proteins that extend unique whisker proteins that are structurally related to podophage GP4 tail fibers, and (iii) a baseplate with long tail fibers resembling a contracted form of T4’s long tail fibers. Sequence conservation analysis of Bas63 structural proteins across ICTV-recognized Felixounavirus’ supports its role as a structural model for Felixounavirus evolution. This study advances the mechanistic understanding of phage architecture and reinforces the structural mosaicism of bacteriophages.

Coliphage N4 is a representative species of the Schitoviridae family of bacteriophages. Originally structurally studied in 2008, the capsid structure was solved to 14 Å to reveal an interesting arrangement of Ig-like decoration proteins across the surface of the capsid. Herein, we present a high-resolution N4 structure, reporting a 2.45 Å map of the capsid obtained via single particle cryogenic-electron microscopy. Structural analysis of the major capsid proteins (MCPs) and decoration proteins (gp56 and gp17) of phage N4 reveals a pattern of interactions across the capsid that are mediated by structurally homologous domains of gp17. In this study, an analysis of the complex interface contacts allows us to confirm that the gp17 Ig-like decoration proteins of N4 are likely employed by the virus to increase the capsid’s structural integrity.

Although over 15,000 unique phage genomes have been sequenced, many isolates remain uncharacterised and accessible only within individual laboratories. The BASEL (BActeriophage SElection for your Laboratory) collection comprises 106 newly isolated and characterised virulent bacteriophages that infect the laboratory strain Escherichia coli K-12 and provide an open resource for phage biology. Here, we used cryo-electron microscopy (cryo-EM) to determine the structure of Bas18, a Dhillonvirus siphophage from the BASEL collection. Bas18 assembles an icosahedral capsid with T=7(d) triangulation. The asymmetric unit contains seven copies of the major capsid protein (MCP; gp09) and one dimeric decoration protein (gp64) bound at the centre of each hexamer. The MCP adopts the canonical HK97 fold but features a distinct insertion between the A and P domains, which we designate as G² and E² loop. The neck assembly consists of the portal, adaptor, stopper and terminator. The helical tail is built from hexameric rings of the tail tube protein, and the tail tip consists of the distal tail protein, hub and central fibre. Despite low sequence similarity, the overall architecture of the neck, tail, and tail tip closely resembles that of bacteriophage T1. In contrast, structural protein sequences are highly conserved (85-99% sequence similarity) across Dhillonviruses indicating a conserved virion architecture within this genus. These results expand the structural knowledge of the BASEL collection and provide a detailed architectural framework for Dhillonvirus phages, contributing to a broader understanding of siphophage structural diversity.