Em grids

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 (link)). 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 (link)).

The electron microscopy was performed at the Multiscale Microscopy Core with technical support from the Oregon Health & Science University (OHSU)/FEI Living Lab and the OHSU Center for Spatial Systems Biomedicine. The samples were imaged using a FEI Talos Arctica system with a FEI Ceta 16M CMOS camera (both from Thermo Fisher Scientific). The samples were prepared by transferring them to Quantifoil EM grids and freezing them in liquid ethane using a Vitrobot prior to imaging. When necessary, the samples were diluted with the sample buffer. The images were processed using the Fiji distribution of ImageJ (NIH, Bethesda, MD, USA) [47 (link)].