Extended Data Fig. 4: Only an antiparallel arrangement agrees with ssNMR data.

ssNMR data (Fig. 1d and Extended Data Fig. 3) provide clear evidence that teixobactin molecules oligomerize upon Lipid II binding and form β-sheets. We confirm oligomerization by several microscopy setups in vitro and in vivo. ssNMR data support that teixobactin molecules feature antiparallel arrangement in oligomers. Unambiguous NMR contacts are shown as continuous blue lines; ambiguous contacts are shown as dashed green lines. For ambiguous contacts, only the contact with the shorter average distance is shown (see Supplementary Tables 5–7). a) Parallel arrangement. This can be ruled out because most intermolecular teixobactin – teixobactin ssNMR contacts would be >>10 Å and the interfacial contact between F1 of teixobactin and MurNAc of Lipid II would be >>20 Å, while the maximum threshold for ssNMR ¹³C-¹³C distance measurements is ~8 Å. Moreover, the depsi-cycles of adjacent teixobactin molecules would clash. b) Shifted parallel arrangement. Steric clashes are avoided, but intermolecular contacts between teixobactin molecules are heavily violated, and contacts between the N-terminus of teixobactin (F1) and Lipid II (MurNAc) remain heavily violated. In addition, the formation of teixobactin – teixobactin hydrogen bonds is compromised. c) Antiparallel arrangement. All Intermolecular and interfacial ssNMR restraints are fulfilled. There are no steric clashes and intermolecular hydrogen bonds can form. An antiparallel arrangement also agrees with the X-ray structure of a teixobactin-sulfate complex in solution⁶⁶. Contacts consistent with antiparallel β-sheets are listed in Supplementary Table T4. Note that some contacts are ambiguous: Contacts with S7α could also be with I11Cα. Note that contacts with I11α would also (even more than with S7α) only be feasible in antiparallel sheets. Likewise, interfacial contacts between MurNAc and F1MeN could also be with Q4γ. Here also, the distance Q4γ - MurNAc – would be >>10 Å in parallel β-sheets.