Folch lipids

BPLE (141101; Avanti Polar Lipids, Alabaster, AL) or Folch lipids (B1502; Sigma Aldrich, St. Louis, MO) were used as is. Lipid stocks (Avanti Polar Lipids) were aliquoted in the following proportions to generate PIP₂-, NTA-, or PIP₂ plus NTA–containing mixtures: DOPC:DOPS:DOPIP₂ (84:15:1 mol%), DOPC:DOPS:NTA (80:15:5 mol%), and DOPC:DOPS:DOPIP₂:NTA (79:15:1:5 mol%). When necessary, trace amounts of the fluorescent lipid probe p-Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (DHPE) was incorporated to a final concentration of 1 mol%. For liposomes, dried lipid mixtures were hydrated in assay buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.5, 150 mM KCl) to a final concentration of 1 mM and extruded through 100-nm polycarbonate membranes (Avanti Polar Lipids). SUPER templates were prepared with PIP₂- or NTA-containing liposomes as previously reported (Pucadyil and Schmid, 2010 (link)). SMrT templates were prepared as described previously (Dar et al., 2015 (link), 2017 ).

Brain-derived Folch lipids (Sigma) were dried under argon gas, dessicated, and rehydrated in buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.5 mM DTT) to a final concentration of 1 mg/ml. To obtain liposomes with different diameters, the lipid mixture was extruded through polycarbon membranes with pore sizes of 0.4, 0.2, 0.1, 0.05, or 0.03 μm (Nucleopore). For liposome co-sedimentation assays, 4 μg of protein was incubated with 0.5 mg/ml liposomes for 30 min at 22°C in a total volume of 50 μl. The reaction was pelleted at 180,000 × g at 22°C for 10 min. Equal volumes of pellet and supernatant fractions were analyzed by SDS-PAGE using NuPAGE Novex Bis-Tris gels (Invitrogen). For in vitro liposome deformation, assay proteins (2.5 μM final concentration) were mixed with 0.125 mg/ml liposomes and incubated for 30 min prior to negative staining EM. Assays were performed two to three times for each protein and with different batches of Folch lipids.