Cryo-EM reveals a conserved baseplate architecture and distinct accessory protein assemblies in mycobacteriophages Claus, Corndog and Mysterious.

Mycobacteriophages, viruses that infect mycobacteria, hold promise as therapeutics against antibiotic-resistant mycobacterial infections. Although thousands of mycobacteriophages have been isolated, high-resolution baseplate structures have been reported for only two, revealing a knowledge gap in understanding phage-host interactions. Most mycobacteriophages are siphophages, possessing four structural components: capsid, connector, non-contractile tail and baseplate. The baseplate plays a critical role in infection by mediating host recognition, adsorption, and genome delivery. Here, we present atomic-resolution cryo-EM baseplate structures of three mycobacteriophages, Claus, Corndog, and Mysterious. These have inverted crown-, barrel-, and cage-like architectures, respectively, but despite their significantly different appearances, the baseplate cores possess a conserved architecture featuring a 6:6:3 stoichiometry of homohexameric tail tube proteins (TTPs), distal tail proteins (DTPs or Dits) and homotrimeric baseplate hub proteins (BHPs). The central core of the baseplate is formed by DTPs and BHPs, with the terminal TTPs making direct proximal contacts with the DTPs and contributing to the overall structure. While the core is morphologically conserved, some domains exhibit notable structural divergence across the three phages. Surrounding the conserved cores are diverse baseplate accessory proteins (BAPs), including upper baseplate proteins (BppUs), central fiber proteins (CFPs) and others, that assemble into distinct peripheral architectures and give rise to two baseplate-opening (or tail tube opening) mechanisms. These architectures reveal a conserved baseplate core complex diversified by BAPs, a feature that may be shared across mycobacteriophage siphophage families.