Challenges and approaches to studying pore- forming proteins

Pore-forming proteins (PFPs) are a broad class of molecules that comprise various fam-ilies, structural folds, and assembly pathways. In nature, PFPs are most often deployed by their host organisms to defend against other organisms. In humans, this is apparent in the immune system, where several immune effectors possess pore-forming activity. Furthermore, applications of PFPs are found in next-generation low-cost DNA sequen-cing, agricultural crop protection, pest control, and biosensing. The advent of cryoEM has propelled the field forward. Nevertheless, significant challenges and knowledge-gaps remain. Overcoming these challenges is particularly important for the development of custom, purpose-engineered PFPs with novel or desired properties. Emerging single -mol-ecule techniques and methods are helping to address these unanswered questions. Here we review the current challenges, problems, and approaches to studying PFPs.