A lipid mixture containing 70 mol-% di-oleyl phosphatidyl choline (DOPC) and 30 mol-% cholesterol in chloroform was dried under a constant nitrogen stream. The lipid film was taken up in 50 mM Tris-HCl pH 7.0, 150 mM NaCl, 5 mM β-mercaptoethanol at a final concentration of 10 mg · ml−1 and stirred overnight at room temperature. After three freeze-thaw cycles (liquid nitrogen, 37°C), the suspension was passed through an extruder to obtain unilamellar ~200 nm liposomes. The extruded liposomes were flash-frozen in liquid nitrogen and stored at −20°C. The liposome suspension was incubated with PLY at a final lipid-to-protein ratio of 1:2 (wt/wt) at 37°C for 30 min. For the preparation of PLY pore complexes, the proteoliposomes were solubilized at a final concentration of 0.56% Cymal-6 at room temperature overnight. Amphipol A8-35 was added in fivefold molar excess and the suspension was incubated for 30 min at room temperature. Detergent was removed by dialysis against 50 mM Tris-HCl pH 7.0, 150 mM NaCl, 5 mM β-mercaptoethanol at room temperature for 72 hr.