Tecnai 12 transmission

For negative staining, purified SCs were diluted to 0.01–0.05 mg/mL concentrations in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.01% DDM, and 1 mM AEBSF) buffer, 5 μL applied to glow-discharged (20 s, 25 mA, Pelco easiGlow) carbon-coated Cu grids (CF300-CU, EMS), incubated for 1 min, and stained with 2% uranyl acetate. Grids were imaged on FEI Tecnai 12 transmission electron microscope operating at 120 kV, with a CCD camera (Gatan BM-Ultrascan).
For cryo-EM, the tripartite SC was used as purified, whereas the purified bipartite SC was mixed with a 5-fold molar excess of purified cyt c_y^{75 (link)}. In all, 2.5 μL of the mixture, containing 3–5 mg/mL proteins, were applied to CFlat holey carbon grids (1.2/1.3–400 mesh or 2/2–300 mesh) (EMS), which were glow discharged (2 min, 25 mA, Pelco easiGlow) before sample application. Grids were blotted for 9 s at force 0 (CFlat-1.2/1.3) or for 3 s at force −5 (CFlat-2/2) and flash-frozen in liquid ethane cooled with liquid nitrogen using FEI Vitrobot Mark IV (25 °C, 100% humidity). Plunge freezing conditions were optimized using FEI Tecnai TF20 TEM operating at 200 kV, equipped with an FEI Falcon II camera.