Deployed by both pathogenic bacteria and host immune systems, pore-forming proteins rupture target membranes and can serve as conduits for effector proteins. Understanding how these proteins work relies on capturing assembly intermediates. Advances in cryoEM allowing in silico purification of heterogeneous assemblies has led to new insights into two main classes of pore-forming proteins: membrane attack complex perforin (MACPF) proteins
and binary toxins. The structure of an immune activation complex, sMAC, shows how pores form by sequential templating and insertion of b-hairpins. CryoEM structures of bacterial binary toxins present a series of transitions along the pore formation pathway and reveal a general mechanism of effector protein translocation. Future developments in time-resolved
cryoEM could capture and place short-lived states along the trajectory of pore-formation.