Reconstitution of Fluc was carried out as described previously (14 (link)). Briefly, 25 mg/mL chloroform stocks of EPLs (Avanti Polar Lipids Inc. #100600C) were dried by evaporation under a stream of nitrogen gas until a thin dried film of lipids appeared. The film was washed with pentane (Sigma-Aldrich #236705-1L). Then, lipids were resolubilized in the required dialysis buffer with 35 mM CHAPS (Anatrace #C316S) for a final concentration of 20 mg/mL lipids. The lipid-detergent mixture was solubilized using a cup-horn sonicator (Qsonica, Newtown, CT) until the sample was transparent. Protein was added to the solubilized lipid-detergent mixture and placed into 3,500 MWCO dialysis cassettes (Thermo Scientific #66330), and then the samples were then dialyzed in the dark at room temperature in 1,000× sample volume with 4 buffer changes every 4 to 12 h. At the end of dialysis, the samples were freeze-thawed to form large paucilamellar vesicles. This involved 3 repetitions of freezing the samples at –80 °C and thawed in a room-temperature water bath. Samples were stored at room temperature, in the dark for the desired amount of time or frozen at –80 °C until used.