Structure-based virtual screening for PLY inhibitors. In order to apply a structure-based virtual screening approach for identifying inhibitors of PLY dimer formation, crystallographic data of PLY monomers were fitted into a cryo-EM map of the pre-pore (Figure ) . This provided a structural model of a PLY dimer (Supplementary Figure ), which led to the identification of potential target sites for small organic molecules (Figure ). The proposed pocket is located directly next to the dimer interface in close proximity to a salt bridge formed by E151 to K288 of the adjacent monomer. The functional relevance of this dimerization hotspot was validated by site-directed mutant of E151Q (Supplementary Figure ). Moreover, the region of the proposed binding site is crucial for large-scale rearrangements upon pre-poreto-pore formation. Using a docking approach about 555,000 molecules were virtually screened, the hit list was filtered according to physicochemical properties and the hit molecules were re-scored by fitting to a pharmacophore model based on frequent interactions. Finally, 10 molecules were selected after clustering for chemical diversity and visual inspection (Supplementary Figure , Supplementary Table. 1).