Subsequent AFM frames of the same area were alternatively recorded from top to bottom and from bottom to top, as indicated by white arrows. Frame time: 4 min, colour scale: 35 nm. (A) Loosely bound to sphingomyelin-rich domains in the phase-separated lipid mixture (DOPC:sphingomyelin:cholesterol, 33:33:33%), the prepore intermediates of disulphide-locked suilysin appear as diffuse streaks. 5 mM of DTT is injected on about 50% completion of the scan. (B) On consecutive scanning, the streaks become more clearly defined as arc-shaped oligomers and complete rings. Towards the top end of the scan, clusters of arc-shaped complexes, mostly in the prepore intermediate (∼10.5 nm high), can be distinguished (Δ). (C) With the scan direction reversed, and the same area scanned again, the cluster of prepore complexes has converted into the pore state (∼7.5 nm high), within ∼2 min. The prepore to pore transition is followed by the ejection of globular features of varying dimensions exceeding 15 nm above the suilysin in the pore state. We interpret these as ejected lipids. (D) These lipids gradually detach from the surface on the pore state suilysin assemblies and can be observed as patches of lipids condensing back onto the membrane. The prepore to pore transition of the suilysin is now complete. (E) After ∼20 min, the surface is almost clear of the ejected lipids. (F) Cross-sectional line profile extracted as indicated (Δ in B–C), illustrating the prepore to pore transition. (G) Cross-sectional line profile extracted as indicated (◊ in D–E), illustrating the lipid ejection and eventual formation of an aqueous pore in the membrane (*).