Reconstitution of ESCRT Complex

We prepared micelles by solubilizing a dried lipid film made from DOPC/DOPS (60/40 mol/mol) at 25 °C in 100 mM NaCl, 20 mM Hepes pH = 7.5, 20 mM CHAPS (3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate hydrate, Sigma-Aldrich) at a total lipid concentration of 12 mM. The following protocol is adapted from^{25 (link)}. In brief, micelles were homogenized by bath sonication and stirring at 25 °C for 1 h before addition of 4 µM Snf7, 2 µM Vps24 and 2 µM Vps2, making sure that the detergent concentration was above its critical micellar concentration after addition of all proteins. The sample was then gradually diluted four-fold over 30 min under agitation at 25 °C and further incubated for 5 h. For cryo-EM, 4 µL of the sample were deposited on glow-discharged Quantifoil R1.2/1.3 300 mesh copper grids and plunge frozen in liquid ethane after a two-sided blot using a FEI Vitrobot.