“…The steps in canonical CDC pore-formation have been well characterised; the proteins are secreted as soluble monomers, monomers bind to and then oligomerise on target membranes to form a ring-shaped prepore complex, the prepore complex then undergoes a concerted conformational change and inserts in the membrane to form a large (25–30 nm) amphipathic β-barrel bilayer-spanning pore ( Figure 1A ; Morton et al, 2019 ). To resolve these steps, point mutations of PFO have been used to investigate kinetically trapped intermediates alongside fluorescent conjugates acting as environmental indicators ( Evans and Tweten, 2021 ; Ramachandran et al, 2004 ), with some intermediates observed by atomic force microscopy (AFM) ( Czajkowsky et al, 2004 ) and electron microscopy (EM) ( van Pee et al, 2017 ) high-resolution imaging. These and other studies have defined the key molecular events and rearrangements required for pore formation.…”