Bacterial surface layers are paracrystalline assemblies of proteins that provide the first line of defense against environmental shocks. Here, we report the 3D structure, in situ localization, and orientation of the S-layer deinoxanthin-binding complex (SDBC), a hetero-oligomeric assembly of proteins that in Deinococcus radiodurans represents the main S-layer unit. The SDBC is resolved at 11-Å resolution by single-particle analysis, while its in situ localization is determined by cryo-electron crystallography on intact cell-wall fragments leading to a projection map at 4.5-Å resolution. The SDBC exhibits a triangular base with three comma-shaped pores, and a stalk departing orthogonally from the center of the base and oriented toward the intracellular space. Combining state-of-the-art techniques, results show the organization of this S-layer and its connection within the underlying membranes, demonstrating the potential for applications from nanotechnologies to medicine.

Department of Life Sciences and Chemistry Jacobs University Bremen 28759 Bremen Germany

Department of Physics and IOM CNR University of Cagliari 09042 Monserrato Italy

Department of Plant Physiology Warsaw University of Life Sciences SGGW 02 776 Warsaw Poland

Department of Plant Physiology Warsaw University of Life Sciences SGGW 02 776 Warsaw Poland; Laboratory of Plant Physiology and Photobiology Department of Life and Environmental Sciences University of Cagliari 09123 Cagliari Italy

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University 12800 Prague Czech Republic

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The cryo-EM structure of the S-layer deinoxanthin-binding complex of Deinococcus radiodurans informs properties of its environmental interactions