Extended Data Fig. 9: Structure properties of the 2TM glycine zipper domain.

(a) helical wheel representation of the SlyB α1 and α2 helices that together form a conserved 2TM glycine zipper domain, and multiple sequence alignment of the corresponding regions of the six different 2TM Gly zipper domain containing proteins commonly identified in the E. coli genome (sequences show Uniprot ID and protein name, as well as MW of the full length proteins; numbering according to SlyB sequence). Hydrophobic and polar side chains are coloured orange sand and white, resp. the conserved Gly or Ala residues in the GXXXG motifs forming the Gly zipper are coloured magenta. Dotted lines connect opposing residues in the α1 - α2 helical packing. (b) Ribbon representation of the 2TM Gly zipper domain as found in the SlyB structures reported in this study. Side chains are shown in stick representation, with N atoms coloured blue, O atoms coloured red and C atoms colour coded according to (a). Shown as pruple, yellow and magenta stick representation are the N-terminal lipid anchor, and a bound LPS and PL molecule in the SlyB protomers. LPS binds the loop connecting α1 and α2 by means of three H-bonds (red dotted lines), two between LPS fatty acids and the G79 and G80 backbone amine, and one between the D-glycosamine-4-phosphate and T82 side chain hydroxyl. In the transmembrane region α1 and α2 pack by the GXXXG knobs-in-hole pattern, with an additional stabilization of three H-bonds connecting the N- and C-terminal region of α1 and α2 resp. (i.e. N60 carbonyl – Q100 side chain amide and the N60 side chain amide with the Q103 side chain amide and the S104 hydroxyl). OM: outer membrane. (c, d) Side and top view ribbon representation of two adjacent SlyB protomers in the SlyB₁₁ oligomer structure (see Fig. 3), with the 2TM domains, lipid anchor, LPS and PL coloured as in Extended Data Fig. 5; and the periplasmic domains coloured blue to red from N- to C-terminus. Secondary structure elements are labelled α1 - α2 and β1 - β6 with A or B superscript for the left or right protomer, resp. Protomers interact by β-sheet augmentation in the periplasmic domain, i.e. strand β1 pairs with β4 of the adjacent SlyB protomer. In the transmembrane region, the protomers interact by non-specific hydrophobic contacts, so that SlyB does not form an obligate oligomer, and can also exist as a stable monomeric or non-circular, low MW oligomeric transmembrane protein. (e) Side-by-side comparison of side and top view cryoEM 2D classes of the OmpC trimer, LMW SlyB:OmpC complexes and SlyB₁₈-OmpC complexes identified in a SlyB – OmpC double affinity purification. Top view classes of LMW SlyB:OmpC complexes did not show a well-resolved binding site for SlyB. (f) Schematic representation of SlyB:OMP nanodomains and side and top view cryoEM 2D classes of SlyB₁₄:BtuB and SlyB₁₂:TSX purified by double affinity purification. Top view classes show the presence of the BtuB or TSX β-barrels encapsulated by a SlyB oligomer of variable protomer number corresponding to the diameter of the enclosed OMP. (left) side view cross section and top view of the cryoEM reconstructions of the SlyB₁₄:BtuB and SlyB₁₂:TSX complexes. The N-terminal lipid anchor, PL, LPS and detergent micelle (M) are coloured slate, magenta, yellow and light blue, resp. β-barrels and SlyB oligomer are separated by a luminal PL bilayer, and SlyB oligomers are surrounded by a LPS ring. Side view classes show the inner core glycans protruding over the rim of the SlyB oligomer, as also seen for SlyB oligomers (Extended Data Fig. 4) and SlyB:BamA complexes (Extended Data Fig. 5). Maps contoured at σ = 3.4 (SlyB₁₂:TSX) or σ = 3.6 (SlyB₁₄:BtuB), with highlighted sections carved at 3.0 Å around the corresponding model coordinates. (g) Species wheel representation of the genomic distribution of the 2TM Gly zipper family (PFAM family PF05433; https://pfam.xfam.org/family/Rick_17kDa_Anti#tabview=tab7). At least 12 discrete protein architectures containing the 2TM Gly zipper domain can be discerned, encompassing at least 4133 sequences originating from 1832 annotated species. Individual genomes hold 1 to 12 2TM Gly zipper containing sequences. The primary occurrence of 2TM Gly domain containing sequences is in Gram-negative phylum proteobacteria, with a smaller fraction in Ascomycete fungi. SP: signal peptide; 2TM: 2TM Gly Zipper domain (green); OmpA: OmpA domain (blue; PFAM: PF00691; cell wall anchoring); Rick_17KDa: 17 kDa outer membrane surface antigen domain (fuchsia; PFAM: PF16998); CVNH: CyanoVirin-N Homology domain (yellow, PFAM: PF08881; glycan binding domain); LysM: LysM domain (magenta; PFAM: PF01476; peptidoglycan binding); β/γ cryst: bacterial β/γ crystallin domain (brown; PFAM: PF00030); RcnB: Nickel/cobalt transporter regulator domain (dark orange; PFAM: PF11776).