Pore formation is central to cholesterol dependent cytolysins’ (CDCs) ability to act as important bacterial virulence factors. Secreted as monomers by Gram-positive pathogens, the toxins self-assemble on the target membrane to form a ring of 30 to 50 subunits. These rings form giant circular pores between 200 to 300 Å in diameter. Each monomer contributes two β-hairpins, termed the TransMembrane Hairpins (TMH1 and TMH2), to the final pore. The current model of CDC pore formation supported by biophysical and cryo-Electron Microscopy (cryo-EM) studies, has demonstrated that the toxins first bind the target membrane via Domain 4. Monomers then self-assemble into a circular ring-shaped oligomer termed the “prepore” complex. The release of the TMH regions from the body of the protein to form wide pores is associated with the vertical collapse of Domains 1/3. It is thought that Domain 2, a twisted sheet connecting Domain 4 and Domains 1/3, experiences substantial structural changes for this vertical collapse to occur. Until recently, the massive conformational rearrangement required to transition from the prepore to pore state had remained unclear. We investigated the vertical collapse employing a combined molecular modelling and structural bioinformatics approach. Systematic analysis of crystallographic CDC structures identified regions and trajectories of inter-domain motions surrounding Domain 2. Critically, the likely swivelling of Domain 2 provides a rationale for the vertical collapse into the final pore. Analysis of available cryo-EM CDC prepore and pore structures further suggests that the prepore-to-pore transition is enabled by coordinated rotations of all domains of the molecule throughout the oligomeric assembly. Furthermore, our study shows that CDC toxins deploy a unique β-barrel architecture in order to form such large pores. Taken together this work provides a new and testable mechanism by which the CDC toxins achieve pore formation.