Talos l120c 120 kv

Three microliter of NDH-1L (for solving the NDH-PQ structure) or NDH-Fd (for solving the NDH-Fd structure) samples at concentration of 12 mg mL⁻¹ were applied to a glow-discharged holey carbon copper or gold grids (GIG, R1.2/1.3, 300 mesh). The grids were blotted for 2 s with blot force of level 2 at 95% humidity and 4 °C, and were plunge frozen into liquid ethane cooled by liquid nitrogen immediately using a semiautomatic plunge device (Thermo Fisher Scientific Vitrobot IV). Sample screen was performed using Talos F200C 200 kV or Talos L120C 120 kV electron microscopes equipped with a Ceta camera (Thermo Fisher Scientific). For structure determination, the micrographs of both NDH-PQ and NDH-Fd were collected using SerialEM software suite^{55 (link)} on a 300 kV Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a K2 camera (Gatan) and a GIF quantum energy filter (20 eV). Total of 1463 images for NDH-PQ and 2090 micrographs for NDH-Fd were acquired using a defocus range between 0.5 and 2.5 μm at 130,000 magnification. The micrographs were exposed for 10 s and dose fractionated into 32 frames, leading to a total dose of 60 e⁻ Å⁻².