Cryo-EM structure and polar assembly of the PS2 S-layer of .

The polar-growing have a complex cell envelope architecture characterized by the presence of a specialized outer membrane composed of mycolic acids. In some , this mycomembrane is further supported by a proteinaceous surface layer or "S-layer," whose function, structure, and mode of assembly remain largely enigmatic. Here, we isolated ex vivo PS2 S-layers from the industrially important and determined its atomic structure by 3D cryo-EM reconstruction. PS2 monomers consist of a six-helix bundle "core," a three-helix bundle "arm," and a C-terminal transmembrane (TM) helix. The PS2 core oligomerizes into hexameric units anchored in the mycomembrane by a channel-like coiled-coil of the TM helices. The PS2 arms mediate trimeric lattice contacts, crystallizing the hexameric units into an intricate semipermeable lattice. Using pulse-chase live cell imaging, we show that the PS2 lattice is incorporated at the poles, coincident with the actinobacterial elongasome. Finally, phylogenetic analysis shows a paraphyletic distribution and dispersed chromosomal location of PS2 in as a result of multiple recombination events and losses. These findings expand our understanding of S-layer biology and enable applications of membrane-supported self-assembling bioengineered materials.