Structure of the hexagonal surface layer on Caulobacter crescentus cells

Abstract

Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and Gram-negative bacteria and most archaea^1-5. S-layers protect cells from the outside, provide mechanical stability and also play roles in pathogenicity. In situ structural information about this highly abundant class of proteins is scarce, so atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified Caulobacter crescentus' sole S-layer protein RsaA, we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. We also solved a 7.4 Å structure of the S-layer through electron cryotomography and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the electron cryotomography map, resulting in a pseudo-atomic-level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. The cellular S-layer atomic structure shows that the S-layer is porous, with a largest gap dimension of 27 Å, and is stabilized by multiple Ca²⁺ ions bound near the interfaces. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with electron cryotomography and sub-nanometre-resolution sub-tomogram averaging.

Figure 1. Arrangement of the Caulobacter crescentus S-layer on cells and stalks.

(A) A tomographic slice of a CB15 C. crescentus cell. The S-layer is continuous between the cell body and the stalk. Inset – a magnified tomographic slice through a S-layer of a stalk. A hexameric lattice with a ~220 Å spacing is seen (see also Movie S1). (B) A magnified tomographic slice of a side view of the cell surface. The S-layer is arranged in two layers and is seen ~180 Å away from the outer membrane of the cell. The outer S-layer lattice is highly inter-connected while the inner domains are ~220 Å apart from each other. (C) A schematic representation of the cell surface.

Figure 2. 2.7 Å X-ray structure of the outer S-layer lattice.

(A) Schematic diagram of the RsaA protein sequence. The N-terminal region of RsaA is a putative LPS binding region (see Figure S2), residues 249-1026 are resolved in the X-ray structure (below) and residues 618-1026 have predicted sequence similarity with Ca²⁺-dependent β-sheet rich proteins (HHPred server 50). (B) On addition of Ca²⁺ ions to RsaA, 2D sheets were obtained with the same appearance and repeat as S-layers on cells. A cryo-ET slice through a reconstituted sheet is shown (see also Movie S2). (C) Crystal structure of RsaA: one monomer of RsaA (see Table S1) shown as ribbon diagram, coloured as a rainbow, N- to C-terminus from blue to red. Two orthogonal orientations are shown with the N- and C-termini of the protein marked. Ca²⁺ ions are shown as grey spheres. The inset shows a region around amino acid 653 where three tightly bound structural Ca²⁺ ions stabilise the fold. (D) Non-crystallographic hexamer of RsaA (forming the asymmetric unit of the crystals) resolved in the X-ray structure, shown in two orthogonal orientations. The central pore is roughly 20 Å wide while the lattice is about 70 Å thick. The N-termini of the six monomers of RsaA point towards the cell surface.

Figure 3. 7.4 Å cryo-ET and sub-tomogram averaging map of the C. crescentus S-layer.

(A) A top view (from outside the cell) of the outer layer of the sub-tomogram averaging map (grey density) with the X-ray structure (blue ribbon) fitted into the density (see also Figure S4). The cryo-EM density is shown at an isosurface threshold level of 2.0 σ away from the mean (red number on the bottom left). The entire hexamer as determined by X-ray crystallography is fitted into the outer S-layer lattice as a rigid body without further adjustments, demonstrating very strong agreement between the X-ray structure determination and cryo-ET. (B) A slice through the outer S-layer lattice with three of the six RsaA molecules from the X-ray hexamer overlaid (see also Figures S4-5 and Movie S6). (C) Side view of the sub-tomogram averaging map shows that the N-terminal RsaA residue 249 in the X-ray structure is located in the short linker between the two domains of the S-layer (see Figures S4E). (D) A single slice through the cryo-ET map, with the fitted RsaA hexamer from the X-ray model overlaid (a clipping plane has been applied at the front and the back). (E) A top view of the hexamer of inner domains of the S-layer. (F) A slice through the inner domain density suggests that it might contain some α-helical secondary structure elements.

Figure 4. The S-layer lattices observed in the X-ray structure and the cryo-ET map are exceptionally similar.

(A) Final refined positions of sub-tomograms are shown plotted back onto a tomogram of a cell stalk with the corresponding refined orientations (see Movie S7). Positions have been coloured from blue (high cross-correlation of alignment) to red (low cross-correlation). One slice of the tomogram is shown with protein density black. (B) The same plot as panel A, except each hexamer position is illustrated with the sub-tomogram average (green volumes). One hexamer is highlighted in blue, and the hexamers directly contacting it are shown in orange. (C) A zoomed view of the hexameric lattice revealed by cryo-ET and sub-tomogram averaging. The 220 Å hexamer:hexamer spacing is highlighted. The central blue hexamer from panel B is replaced by one copy of the X-ray hexamer. (D) The RsaA lattice formed in the crystals through crystal packing is the same as the S-layer lattice observed on cells through cryo-ET. The 220 Å hexamer:hexamer repeat is shown. One hexameric, trimeric and dimeric interface, each have been highlighted with a black hexagon, a triangle and a line, respectively. In conclusion, both X-ray crystallography and cryo-ET used in this study show essentially the same lattice and arrangement of RsaA in C. crescentus S-layers. Also see Movies S4, 7. (E) Atomic structure of the S-layer. (F) A slice through an in vitro assembled RsaA sheet (see Movie S2) for comparison at the same scale as panel E.