The concept of self-assembly is one of the most promising strategies for the creation of defined nanostructures and therefore became an essential part of nanotechnology for the controlled bottom-up design of nanoscale structures. Surface layers (S-layers), which represent the cell envelope of a great variety of prokaryotic cells, show outstanding self-assembly features in vitro and have been successfully used as the basic matrix for molecular construction kits. Here we present the three-dimensional structure of an S-layer lattice based on tetrameric unit cells, which will help to facilitate the directed binding of various molecules on the S-layer lattice, thereby creating functional nanoarrays for applications in nanobiotechnology. Our work demonstrates the successful combination of computer simulations, electron microscopy (TEM), and small-angle X-ray scattering (SAXS) as a tool for the investigation of the structure of self-assembling or aggregating proteins, which cannot be determined by X-ray crystallography. To the best of our knowledge, this is the first structural model at an amino acid level of an S-layer unit cell that exhibits p4 lattice symmetry.