Preformed liposomes were incubated with PLY (1 mg · ml−1) at room temperature for 30 min to obtain prepores, or at 37°C for 30–180 min to obtain pores. For cryoET the liposomes were diluted 1:3 with buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.0, 5 mM β-mercaptoethanol) and then 1:1 with 6 nm gold particles conjugated with protein A (Aurion) as fiducial markers. Samples of 3 µl were applied to glow-discharged Quantifoil EM grids (R2/2, Cu 300 mesh), excess liquid was blotted off with filter paper (Whatman #4) and samples were vitrified by plunge-freezing into liquid ethane (Dubochet et al., 1988). Single-axis tilt series were typically collected from −62° to +62° at 2° increments and 3–4 µm underfocus with a total electron dose of 60–80 e- · Å-2, on a Tecnai Polara electron microscope equipped with a field emisson gun operating at 300 kV (FEI, Hillsboro, OR), a post-column energy filter (GIF Quantum, Gatan) and a K2 summit direct electron detector (Gatan). Images were recorded in counting mode with a pixel size of 0.35 nm. Tilt series were CTF-corrected, binned 2 × 2 and aligned. Tomographic volumes were generated by weighted back projection in IMOD (Kremer et al., 1996).