MACPF and CDC proteins form two branches of a superfamily and assemble into large transmembrane pores by a similar mechanism. For the more extensively studied CDCs, it is established that monomers first assemble into a prepore on the membrane. Conversion to the pore involves straightening of the central β−sheet of each subunit, collapse of the prepore structure towards the membrane and refolding of two α-helical regions into transmembrane β-hairpins TMH1 and TMH2. The molecular details of MACPF pore formation remain to be characterised. We determined cryo EM structures of three disulphide bond-trapped prepore intermediates of MACPF protein pleurotolysin. These prepore structures reveal that the oligomers can assemble andthe core sheet can open almost fully without the TMH regions inserting into the membrane. Molecular modelling and flexible fitting allowed us to generate a potential molecular trajectory of β-sheet opening during pore formation. This opening requires mobility of TMH2, and may be triggered by a displacement of a conserved helix-turn-helix motif at the top of TMH2 region. MACPF sheet straightening leads to formation of a nascent β-barrel that templates top down assembly of TMH1 and TMH2 into a transmembrane pore, a mechanism that may be conserved across the whole superfamily.