C flat 1.2 1 3 300 mesh copper grids

Three microliters of purified sample was applied onto freshly glow-discharged (15 mA for 45 s in a PELCO easiGlow system) C-flat 1.2/1.3 300-mesh copper grids (Science Services GmbH) at a final concentration of 1.2 mg/ml. Samples were blotted for 4.5 s with a blot force of 20 (595 Whatman paper) at 4°C in 100% humidity and plunge-frozen in liquid ethane by using a FEI Vitrobot Mark IV. Cryo-EM data were collected automatically using EPU software (Thermo Fisher Scientific) on a Titan Krios G3i microscope operating at 300 kV and equipped with a K3 detector operating in electron counting mode. Movies were acquired at a nominal magnification of ×105,000, resulting in a pixel size of 0.837 Å. Each movie was recorded for 3 s and subdivided into 60 frames. Electron flux rate was set to 14 e⁻ per pixel per second at the detector, resulting in an accumulated exposure of 60 e⁻/Ų at the specimen. An energy filter slit width of 30 eV was used, and a 70-μm C2 and 100-μm objective aperture were inserted during acquisition. A total of 6229 movies at a defocus range of −1 to −3 μm were collected.

Three microliters of purified complex I at a final concentration of 3.6 mg/ml (DDM-solubilized) or 1.5 mg/ml (LMNG-solubilized) was applied onto freshly glow-discharged (15 mA for 45 s in a PELCO easiGlow system) C-flat 1.2/1.3 300 mesh copper grids (Science Services GmbH). Samples were blotted for 4 s with blot force 20 (595 Whatman paper) at 4°C in 100% humidity and plunge-frozen in liquid ethane with a FEI Vitrobot Mark IV. CryoEM data were collected automatically using EPU software (Thermo Fisher Scientific) on a Titan Krios G3i microscope at 300 kV, equipped with a K3 detector (Gatan) operating in electron counting mode. Movies were acquired at a nominal magnification of ×105,000, resulting in a pixel size of 0.837-Å. Each movie was recorded for 2.1 s and subdivided into 45 frames. The electron flux rate was set to 15 e⁻ per pixel per second at the detector, resulting in an accumulated exposure of 45 e⁻/Ų at the specimen. An energy filter with a slit width of 30 eV was used, and a 70 μm C2 and a 100 μm objective aperture were inserted during acquisition. A total of 1996 movies at a defocus range of −1 μm to −3.5 μm were collected for complex I solubilized in DDM and 6570 movies at a defocus range of −0.5 to −3.0 μm for complex I solubilized in LMNG. An overview of the data collection statistics is shown in table S3.