Fig. 7. TS structures formed during BamA folding in PG. (a and b) Two-dimensional topology diagram of BamA highlighting residues that form native-like (blue), unfolded-like (red), or non-native interactions (gray) in the transition state structure of BamA in the fast (Φ_F1-PG) (a) and slow (Φ_F2-PG) (b) pathways. Φ_F ranges used for each color code are indicated below (boxed). β-Strand numbers are annotated in (a) in blue. Residues with ΔΔG⁰_F ≤ −0.09 kcal mol⁻¹ or ≥ +0.09 kcal mol⁻¹ (ΔΔG⁰_F > 10% significance) are shown as uppercase single-letter abbreviations. Residues with ΔΔG⁰_F ≤ −0.05 kcal mol⁻¹ or ≥ +0.05 kcal mol⁻¹ (ΔΔG⁰_F between 5–9.99%) are in lowercase, italics. To minimize errors in the analyses, mutants with ΔΔG⁰_F between ± 0.0499 kcal mol⁻¹ were excluded from the topology diagram. Local zones in β-strands which show several native-like interactions are considered folded in the transition state, and are highlighted in light blue casing. Complete data in Fig. S30. The two transition states in the fast (Φ_F1-PG) (a) and slow (Φ_F2-PG) (b) pathways show similar assembly structures with the C-terminal strands largely folded. Differences in both pathways largely originate from the correct assembly of G⁶³¹ on β11. In Φ_F1-PG, native-like interactions established by this residue facilitate rapid assembly of BamA by promoting the formation of more native-like interactions at the N-terminal strands (a). However, the non-native interactions formed by G⁶³¹ in Φ_F2-PG (b) slow BamA assembly in this pathway, which is evident from the several residues distributed across β5–β10 that remain unfolded in the transition state (shown in red) or form non-native interactions (β3–β6; shown in gray). Both TS structures exhibit directional C → N formation of the BamA barrel, similar to the proposed in vivo assembly mechanism. (c) Illustrated here are the TS structures we captured in PG. Structures (generated using PyMOL) are presented on the left, and their cartoon diagrams on the right extreme. In PG-rich membranes (pale purple), we obtain a more structured TS for nBamA, with similar native-like interactions at several loci in both fast (i) and slow (ii) pathways. Both pathways share similar structured regions, involving β7–β8, β10–β12, and β13–β16 (insets). Assembled strands also extend from β6–β13 in the fast pathway (i; left insets).