Lipid chloroform stocks

Liposomes were prepared as previously described (McDonald et al., 2015 (link)). Lipid chloroform stocks (Avanti Polar Lipids, Alabaster, AL) were mixed at the desired ratios, vacuum dried into a thin film in a glass tube, and resuspended at 1 mg/ml in 20 mM Tris, pH 8.0, 150 mM NaCl buffer with vortexing. The resulting liposome mix was freeze-thawed 10x and passed through an 800 nm liposome extruder (Avanti Polar Lipids, Alabaster, AL). 100 μl liposomes were mixed with 100 μl purified Rga7 or Rng10 protein at the indicated concentrations (or 20 μg when not indicated) and incubated for 15 min at room temperature. Bound liposomes were centrifuged at 150,000 g for 15 min and supernatant (unbound) and pellet (bound) fractions were separated and run on an SDS-PAGE for Coomassie blue detection.
Giant unilamellar vesicles were formed as previously described (McDonald and Gould, 2016a (link)). Chloroform lipid stocks of the desired composition were evaporated on ITO-coated glass coverslides. Coverslides were assembled into a 1 mm thick chamber filled with a 20 mM HEPES, pH 7.4, 500 mM sucrose solution. A 10 Hz, 2.5 V sine current was passed across the chamber for 2 h to electroform the GUVs.

Nanodiscs (NDs) were assembled according to established protocols (52 (link), 53 (link)). In short, lipid chloroform stocks (Avanti Polar Lipids, Alabaster, AL) were dried under nitrogen flow to obtain a lipid film and stored under vacuum overnight. ΔHis MSP1D1 and the appropriate amount of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) solubilized in 60 mM Na-cholate were mixed together in 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA. The scaffold-to-lipids molar ratio was calculated from geometrical considerations. A quantity of 20% w/v of previously washed (methanol, water, buffer twice) Biobeads SM-2 (BioRad, Hercules, CA) was added and the mixture incubated at room temperature overnight. The Biobeads were removed by centrifugation and once again 20% w/v was added for an additional 4–5 h. Finally, they were purified by SEC on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) equilibrated with 20 mM sodium phosphate pH 7.4, 50 mM NaCl using an Äkta pure device (GE Healthcare) run at 1 mL/min. The quality of ND preparation was evaluated by the SEC chromatogram as well as by DLS (Nicomp system; Particle Sizing Systems, Port Richey, FL). NDs were concentrated to the desired molarity using a Vivaspin centrifugal device of 10 kDa MWCO.