Extended Data Figure 2. VmFtsA forms antiparallel double filaments upon binding VmFtsN^1-29.

a, Schematic overview of EcFtsN. b, SPR equilibrium response titration of VmFtsN^1-29 binding to immobilised VmFtsA^1-396. Binding affinity is about three fold lower than for the EcFtsA^1-405-EcFtsN^1-32 interaction (Figure 2b). c, VmFtsA^1-396 titration into VmFtsN^1-29-C-Atto 495. Data were fitted with a two step model, with transitions being indicative of FtsN binding and polymerisation (panel d). A representative quadruplicate is shown. K_ds are given as mean ± SEM from five independent experiments. d, Weight-averaged sedimentation coefficients of a VmFtsA^1-396 titration into VmFtsN^1-29-C-Atto 495 by FDS-AUC shows that VmFtsN^1-29 is part of higher order FtsA polymers. Data were fitted to a two step model, recapitulating the FP data in panel c. e, Co pelleting assay of VmFtsN^1-29 titrated into VmFtsA^1-396 indicates that VmFtsN^1-29 induces FtsA polymerisation. A representative SDS-PAGE gel is shown. Given are mean ± sd (black lines) of technical duplicates (white dots). P: pellet, S: supernatant. f, Negative stain electron micrographs of VmFtsA with and without VmFtsN^1-29 on supported lipid monolayers. VmFtsA forms “mini-rings” in the absence of FtsN and double filaments at ten fold molar excess of VmFtsN^1-29. Two independent grids were examined per condition. Scale bar, 50 nm, 20 nm (inset). g, Multiple sequence alignment of 245 FtsN sequences comprising cytoplasmic and transmembrane domains. EcFtsN^1-32 and VmFtsN^1-29 sequences are highlighted in bold. h, Mapping of the FtsA-interacting region in EcFtsN^1-32 using the lipid monolayer assay. EcFtsN^1-32 mutants were in ten fold molar excess of FtsA. In contrast to EcFtsN^1-32, EcFtsN^{1-32, D5N} and a scrambled version of the EcFtsN^1-32 peptide²⁴ did not induce FtsA double filaments. EcFtsN^4-26 and EcFtsN^{1-32, ΔRK1} led to formation of fewer double filaments. At least two independent grids were examined per condition. Scale bar, 20 nm. i, Summary of EcFtsN^1-32 peptides. Mutations are highlighted in bold. Equilibrium dissociation constants (K^eq_d) are given as mean ± SEM (n ≥ 2 for each construct). For weak binders the maximum response was fixed during fitting, hence these are only approximate values as indicated by asterisks. ND: not determinable. The predominant higher order polymer observed in the monolayer assay is given in the “EM” column. Note that EcFtsN^4-26 and EcFtsN^{1-32, ΔRK1} still lead to formation of a few FtsA double filaments.