Structural basis of virion assembly in the Mycobacterium tuberculosis-infecting mycobacteriophage SWU1

The rising threat of drug-resistant tuberculosis has renewed interest in bacteriophage therapy as an urgently needed alternative. Rational engineering of these viruses, however, requires an atomic-level understanding of virion architecture. Mycobacteriophage SWU1 belongs to subcluster A2, one of the few mycobacteriophage lineages known to infect Mycobacterium tuberculosis (M. tuberculosis). Using single-particle cryogenic electron microscopy (cryo-EM), we resolved essentially the entire SWU1 virion architecture by localized reconstruction of four modules, the capsid, portal vertex, tail and baseplate, at resolutions of 3.62 [A], 3.51 [A], 3.70 [A] and 3.22 [A], respectively. The T=7 capsid comprises 415 copies of an HK97-fold major capsid protein and lacks any decoration protein in density. The 12-fold portal reconciles its C5-C12 mismatch with the capsid vertex through defined, non-equivalent subunit contacts. The baseplate carries a 13-fold tail-fiber ring on a 6-fold hub, a geometry without precedent among characterized phages, established here by symmetry-free reconstruction. These findings provide the critical insights into the assembly and infection of SWU1, laying a structural foundation for the future optimization of phage-based anti-TB therapeutics.