The alpha-PFT, Haemolysin E, HlyE or ClyA of 536 aas. A peptide derived from the putative transmembrane domain in the tail region of hemolysin E (aas 88-120) assembles in phospholipid membrane and exhibits lytic activity to human red blood cells (Yadav et al., 2009). Residues important for insertion and activity have been identified (Ludwig et al., 2010).  An unusual assembly pathway has been proposed (see family description; Fahie et al. 2013). The pore can be blocked by PAMAM dendrimers (Mandal et al. 2016).  The C-terminus directs pore formation and function (Sathyanarayana et al. 2016).  Similar in structure to Cry6Aa (TC# 1.C.41.2.1) although sequence similarity could not be discerned (Dementiev et al. 2016 and unpublished results).  The C-terminal domain is not directly involved in the pore structure, but is not a passive player in pore formation as it plays important roles in mediating the transition through intermediary steps leading to successful pore formation in a membrane (Sathyanarayana et al. 2016). Transmembrane oligomeric intermediates or "arcs" probably form stable proteolipidic complexes consisting of protein arcs with toroidal lipids lining the free edges (Desikan et al. 2017). High-resolution cryo-EM structures revealed that ClyA pore complexes can exist as oligomers of a tridecamer and a tetradecamer, at estimated resolutions of 3.2 Å and 4.3 Å, respectively. The 2.8 A cryo-EM structure of a dodecamer dramatically improves the existing structural model. Structural analysis indicates that protomers from distinct oligomers resemble each other, and neighboring protomers adopt a conserved interaction mode. A stabilized intermediate state of ClyA during the transition process from soluble monomers to pore complexes was identified. Even without the formation of mature pore complexes, ClyA can permeabilize membranes and allow leakage of particles less than ~400 Daltons. In addition, ClyA forms pore complexes in the presence of cholesterol within artificial liposomes (Peng et al. 2019). The mechanism of pore formation has been reviewed (Sathyanarayana et al. 2020). Maurya et al. 2022 described how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. This and other pore forming toxins have been reviewed (Gupta et al. 2023).  A cholesterol binding motif in the membrane-inserted helix of ClyA is present.  Distinct binding pockets for cholesterol are formed by adjacent membrane-inserted helices as revealed in MD simulations. Cholesterol appears to play a dual role by stabilizing both the membrane-inserted protomer as well as oligomeric intermediates. Molecular dynamics simulations and kinetic modeling studies suggest that the membrane-inserted arcs oligomerize reversibly to form the predominant transmembrane oligomeric intermediates during pore formation (Sathyanarayana et al. 2021).