Structure of the hexagonal surface layer on Caulobacter crescentus cells

Abstract

Many prokaryotic cells are encapsulated by a surface layer (S-layer) that consists of repeating units of S-layer proteins (SLPs). S-layers protect cells from the outside, provide mechanical stability, and also play roles in pathogenicity. In situ structural information on this highly abundant class of proteins is scarce, therefore atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified C. crescentus’ sole SLP RsaA we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. Next, we solved a 7.4 Å structure of the S-layer through electron cryotomography (cryo-ET) and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the cryo-ET map, resulting in a pseudo-atomic level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with cryo-ET and sub-nanometre resolution sub-tomogram averaging. The cellular S-layer atomic structure shows that the S-layer is porous, with the largest gap dimension being 27 Å, and is stabilised by multiple Ca2+ ions bound near interfaces.

A Bacterial Surface Layer Protein Exploits Multi-step Crystallization for Rapid Self-assembly

“…RsaA consists of two domains, both of which are necessary for successful S-layer assembly in vivo (Figure 1A) (10, 11). The N-terminal domain, here defined as RsaA 1-246 , is responsible for anchoring the protein to the cell surface via a non-covalent interaction with specific sugar moieties of the lipopolysaccharide (LPS) outer membrane (11–13).…”

“…The N-terminal domain, here defined as RsaA 1-246 , is responsible for anchoring the protein to the cell surface via a non-covalent interaction with specific sugar moieties of the lipopolysaccharide (LPS) outer membrane (11–13). In contrast, the C-terminal region is responsible for making the inter-molecular contacts necessary to form the physiological S-layer lattice (10). A recent 2.7 Å resolution crystal structure revealed that the C-terminus, RsaA 249-1026 , consists of a series of beta-strands arranged in an L-shape and connected by loops interspersed with calcium-binding Repeat-in-Toxin (RTX) motifs (Figure 1A,B) (10, 14).…”

“…In contrast, the C-terminal region is responsible for making the inter-molecular contacts necessary to form the physiological S-layer lattice (10). A recent 2.7 Å resolution crystal structure revealed that the C-terminus, RsaA 249-1026 , consists of a series of beta-strands arranged in an L-shape and connected by loops interspersed with calcium-binding Repeat-in-Toxin (RTX) motifs (Figure 1A,B) (10, 14). Docking this crystal structure into a 7.4 Å resolution electron cryo-tomographic reconstruction of the repeating hexameric unit of the native S-layer lattice confirmed the identity of the side chains coordinating the physiological S-layer crystal contacts (Figure 1A and S1) (10).…”

Lipid Anchoring of Archaeosortase Substrates and Mid-Cell Growth in Haloarchaea

“…First, even though there are examples of bacterial species with S-layer that carry out peptidoglycan cellwall synthesis at mid cell (6), their new S-layer material is inserted as patches distributed all around the cell (35,36). Second, lack of conservation in the protein architecture between archaeal and bacterial S-layers argue that they may have emerged independently of each other (8,37).…”

A guided approach for subtomogram averaging of challenging macromolecular assemblies

“…Multiple copies of the biological complex of interest can be identified within reconstructed tomograms, and 3D "subvolumes" can be extracted and averaged together in a process called subtomogram averaging (STA) [12][13][14][15][16][17][18][19][20] to obtain better resolved 3D reconstructions of the complex of interest. Notably, aspects of single-particle image processing have been incorporated into STA processing packages 18,19,21 , and combined with improved 3D-contrast transfer function (CTF) estimation and missing-wedge compensation 20,[22][23][24] to achieve reconstructions in the sub-nanometer resolution regime 9,20,[25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] . Notably, high-resolution (3.9-3.1Å) reconstructions of the HIV-1 capsid-SPI region of the viral Gag protein has been determined using cryo-ET 20,23,40 , further emphasizing the promise of this technique in obtaining high-resolution structural information of complexes in situ.…”