stepwise visualization of membrane pore formation by suilysin a bacterial cholesteroldependent cytolysin membrane attack complexperforincholesteroldependent cytolysin macpfcdc proteins constitute a major superfamily of poreforming proteins that act as bacterial virulence factors and effectors in immune defence upon binding to the membrane they convert from the soluble monomeric form to oligomeric membraneinserted pores using realtime atomic force microscopy afm electron microscopy em and atomic structure fitting we have mapped the structure and assembly pathways of a bacterial cdc in unprecedented detail and accuracy focussing on suilysin from streptococcus suis we show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane the resulting ringshaped pores and kinetically trapped arcshaped assemblies are all seen to perforate the membrane as also visible by the ejection of its lipids membrane insertion requires a concerted conformational change of the monomeric subunits with a marked expansion in pore diameter due to large changes in subunit structure and packing