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Abstract

Surface layers (S-layers) are two-dimensional (2D) crystalline lattices that frequently coat prokaryotic cells, playing a crucial role in protection, maintaining cellular integrity, and mediating environmental interactions. However, the molecular landscape of these abundant proteins has remained underexplored due to a lack of structural data. By employing AlphaFold2multimer together with planar symmetry constraints in a workflow validated by electron cryomicroscopy structure determination, we have elucidated the lattice structures of over 150 S-layers from diverse archaea and bacteria. Our findings unveil a multifaceted evolutionary landscape for S-layer proteins, highlighting key differences in the evolution of bacterial and archaeal S-layers. Our study allows us to discover underlying patterns in S-layer structure, organisa-tion, and cell anchoring mechanisms across the prokaryotic tree of life, deepening our understanding of the intricately complex microbial cell surfaces, which appear to have evolved proteinaceous S-layers independently on multiple occasions. This work will open avenues for rational manipulation of prokaryotic cellular interactions in multicellular microbiomes, as well as for innovative 2D biomaterial design.