Structure of the assembled 2D bacterial S-layer of SbsC

Surface layers (S-layers) are monomolecular crystalline arrays forming the outermost component of the cell wall of many bacteria and archaea. Due to their potential to form regular crystalline arrays with pores of defined size and shape they can be used in biotechnology, molecular nanotechnology and biomimetics. Little information is available about the specific function(s) of S-layers. In spite of their biological importance for the functionality of prokaryotic cells, high resolution structural information about S-layer proteins is very scarce. A number of 3D reconstructions of negatively stained monolayer self-assembly products using Electron Microscopy (EM) were performed, yielding structural information to approx. 17 Ang. Recently, the projection map using cryo-EM of the S-layer protein SbpA to 7Å was reported. We determined the first atomic resolution structure of a bacterial S-layer protein. Since S-layer proteins exhibit the tendency to self-assemble into 2D crystalline arrays, we had to use N- and C-terminal deletion mutants in order to obtain 3D crystals for structure determination. The structures of the assembly deficient mutants rSbsC (31-844) and rSbsC (31-443) (an S-layer protein from Geobacillus stearothermophilus) show an interesting novel fold and an assembly of domains linked by flexible linkers, but do not reveal how the SbsC protein is able to self-assemble into an ordered 2-D lattice. In order to obtain structural information on the domains involved in the assembly process, crystals of the C-terminal part of the SbsC protein were obtained and diffract to 2.2 Å. The structure determination is under way. The aim of this project is the structural characterization of the 2D crystalline arrays formed by the S-layer protein SbsC using a combination of cryo-EM and X-ray crystallography. The 3D reconstruction of 2D crystals formed by the whole SbsC protein will be studied by Cryo-EM. Using 3D reconstruction techniques it will be possible to gain information about the 2-D lattice at a resolution of approx. 8 Ang, which would enable recognition of individual domains. Fitting of the atomic resolution structures obtained by X-ray crystallography into the electron density obtained from 3D reconstruction will give us an insight into the formation of the 2-D lattice.