A new age in structural S-layer biology: Experimental and in silico milestones.

Surface (S-) layer proteins, considered as the most abundant proteins in nature, perform diverse and essential biological roles in many bacteria and most archaea. Their functions range from providing structural support, maintaining cell shape, and protecting against extreme environments to acting as a cell surface display matrix for biologically active molecules, such as S-layer protein-bound glycans, which facilitate interspecies interactions and cellular communication in both health and disease. The intricate, symmetric, nanometer-scale patterns of S-layer lattices have long fascinated structural biologists, yet only recent methodological advances have revealed detailed molecular insights. These advances include a deeper understanding of domain organization, cell wall-anchoring mechanisms, and how nascent proteins are incorporated into existing lattices. Significant progress in sample preparation and high-resolution imaging has led to the precise structural characterization of S-layers across various bacterial and archaeal species. Furthermore, the advent of deep learning-based structure prediction has enabled modeling of S-layer proteins in several largely uncultured microbial lineages. This review summarizes major achievements in S-layer protein structural research over the past 5 years, presenting them with a typical workflow for the experimental structure determination. For the first time, it also explores recent breakthroughs in computational S-layer modeling and offers an outlook on how in silico methods may further advance our understanding of S-layer protein architecture.