The three-dimensional (3D) structure of one surface protein layer from Bacillus brevis 47, the middle wall (MW) layer, has been reconstructed from tilted-view electron micrographs after correlation averaging to a resolution of 2 nm. The MW layer has p6 symmetry with a center-to-center spacing of 18.3 nm and a minimum thickness of 5.5 nm. The reconstruction reveals a distinct domain structure: the heavier domain of six monomers jointly forms a massive core centered at the sixfold symmetry axis, and lighter domains interconnect adjacent unit cells. In addition, the larger domains collectively form a pore by making contact with each other towards the inner surface, while the smaller domains establish a second connectivity towards the outer surface of the S layer. The MW layer of B. brevis resembles the S layer of Acetogenium kivui in various aspects: they have very similar lattice parameters and highly reminiscent 3D structures; the pores penetrate through the whole core and appear to determine the porosity of the S layers.