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Two-dimensional bacterial surface layer protein crystals (S-layers) are the most commonly observed cell surface structures in prokaryotic organisms (bacteria and archaea). Isolated S-layer proteins have the intrinsic tendency to self-assemble into two-dimensional arrays in suspension and at various interfaces. Basic research on the structure, genetics, chemistry, morphogenesis and function of S-layers has led to a broad spectrum of applications in molecular nanotechnology and biomimetics. The possibility to change the natural properties of S-layer proteins by genetic manipulation opens new ways for the tuning of their structural and functional features. Functionalised S-layer proteins that maintain their propensity for self-assembly have led to new affinity matrices, diagnostic tools, vaccines or biocompatible surfaces, as well as to biological templating or specific biomineralisation strategies at surfaces.