Capsid structure of phage SPO1 reveals novel minor capsid proteins and insights into capsid stabilization.

SPO1-related bacteriophages are promising candidates for phage therapy. We present the 3.0 Å cryo-electron microscopy (cryo-EM) structure of the SPO1 capsid with a triangulation number T = 16, enabling the construction of an atomic model comprising the major capsid protein and three types of minor capsid proteins: gp29.2, gp2.7, and gp36.3. These minor capsid proteins adopt novel folds. They might stabilize the capsid and determine its curvature. Gp29.2 monomers contain a three-blade propeller fold and are located at the 3-fold and quasi-three-fold axes. Gp2.7 forms pentamers atop pentameric capsomers, while gp36.3 binds to the capsid's inner surface, forming star-shaped structures increasing connections between pentameric and hexameric capsomers. The surface exposed regions of gp29.2 and gp2.7 make SPO1 of interest as a nanocage for phage display. Our findings advance the understanding of capsid architecture, stabilization, and local curvature determination for SPO1-related bacteriophages.