Cryo-EM sample vitrification and imaging

Isolated mitochondria were suspended at a concentration of 0.1 mg/mL in 2 mL respiration buffer with 5 mM sodium pyruvate and 1 mM L-malate. At the collection times indicated, 5 µL samples were pipetted from the mitochondrial suspension and deposited on Quantifoil R2/2 Holey Carbon grids that had been plasma-cleaned for 20 s using a Fischione Instruments model 1020 plasma cleaner. Grids were blotted to thin the water layer, and subsequently plunged into liquid ethane at room temperature using a manual plunge-freezing device (Michigan State University Physics Machine Shop). Grids were then transferred and stored in liquid nitrogen until imaging. Data for the 75 µM CaCl₂ experiments were collected in the cryo-EM facility at the University of Pittsburgh School of Medicine using an FEI Polara G2 cryo-electron microscope with a field emission gun operating at 300 kV at nominal magnification of 9400× with a post-column magnification of 1.4× to obtain a ~ 12–10 Å/pixel resolution. Images were recorded on a FEI Falcon 3 direct electron-detecting camera at an electron dose of 0.2 e⁻/Ų. Data for the 100 µM CaCl₂ experiments were collected in the cryo-EM facility at the University of Pittsburgh School of Medicine using a FEI TF20 cryo-electron microscope with a field emission gun operating at 200 kV. The images were collected using a nominal magnification in the range of 5000× on a TVIPS XF416 CMOS camera with a post-column magnification of 1.4× to obtain a 22 Å/pixel resolution. Data for the ± 1 µM FCCP condition at 50 µM CaCl₂ were collected in the cryo-EM facility on Michigan State University using a Talos Arctica outfitted with a Falcon 3 direct electron-detecting camera. The field emission gun was operating at 200 kV using a magnification of 22,000× at an electron dose of 2.03 e⁻/Ų to obtain a 4.69 Å /pixel resolution. Grids were blotted for 5 s before vitrification in liquid ethane using a Vitrobot Mark IV System from Thermo Fisher Scientific. At these magnifications, the electron dose (e⁻/Ų) is low enough to avoid significant sample destruction.