We applied 5 μl of liposomes in PBS (2 mg/ml, ~80 nm diameter by DLS) to a holey grid, which had been previously treated by glow discharged in an oxygen plasma chamber. The cryo–electron microscopy (EM) sample was vitrified using liquid ethane as the cryogen. The frozen sample was transferred into a precooled cryo-transfer holder to maintain a low temperature. The image was acquired on a FEI Tecnai G2 Sphera microscope operated at 200 keV using a Gatan UltraScan 1000 UHS 4 MP charge-coupled device camera. All three lipids (U16, T32, and T36) can form mostly unilamellar liposomes by lipid film hydration and extrusion through polycarbonate membranes (fig. S1, B to D).

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