K3 bioquantum camera

An aliquot of 3.5 μL of purified EV-F4 full capsids at a concentration of 0.3 mg/mL was applied to Quantifoil R2/1 grids coated with 0.2 mg/mL of graphene oxide and previously glow discharged with a negative polarity at 15 mA for 15 s using a PELCO easiGlow. Samples were blotted with Whatman filter paper for 2.5 s before plunged into liquid ethane cooled with liquid nitrogen in a FEI Vitrobot MkIV. Vitrified samples were imaged using the FEI Titan Krios at 300 kV with a Gatan K3 Bioquantum camera and a nominal magnification of 105 kx. Movie frames were collected with a total dose of 41 e/Ų over 30 frames with a final pixel size of 0.857 Å.

To prepare the cryo-EM sample, 3 μl purified complex was applied onto the amorphous alloy film grid (M024-Au300-R12/13)^{45 (link)} or 100 Holey Carbon film (Au, 300 mesh, N1-C14nAu30-01), glow-discharged by the easiGlow™ Glow Discharge Cleaning System (PELCO, USA) at 15 mA for 45 s. At 4 °C and 95% humidity, the sample was waited for 3 s and blotted for 2 s. Grids were plunge-frozen in liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific) and then stored in liquid nitrogen until checked. Movies were collected on a 300 kV Titan Krios Gi3 microscope (Thermo Fisher Scientific FEI, the Kobillka Cryo-EM Center of the Chinese University of Hong Kong, Shenzhen). The raw movies were recorded by a Gatan K3 BioQuantum camera at a magnification of 105,000, and the pixel size was 0.83 Å. Inelastically scattered electrons were excluded by a GIF Quantum energy filter (Gatan, USA) using a slit width of 20 eV. The movie stacks were acquired with the defocus range of −1.1 to −2.0 micron with a total exposure time of 2.5 s fragmented into 50 frames (0.05 s/frame) and with a dose rate of 21.2 e/pixel/s. Automated single-particle data acquisition was performed using SerialEM 3.7.

To prepare the cryo-EM grid for purified Na⁺-NQR, 2.7 μL of the enzyme solution (50 µM) was loaded onto a glow-discharged Quantifoil Cu R1.2/1.3 and blotted by Vitrobot IV followed by vitrification with liquid ethane. To obtain samples with bound aurachin D-42 (abbreviate as “Na⁺-NQR^AD42”) or korormicin A (abbreviate as “Na⁺-NQR^KA”), the enzyme and inhibitor were mixed at a 1:10 molar ratio and incubated for 10 seconds before loading on a grid. Cryo-EM movies were automatically acquired using a Titan Krios electron microscopy (Thermo Fisher, USA) equipped with K3 BioQuantum camera (Gatan, United States) using SerialEM software^{34 (link)}. The cryo-EM movies were collected at a nominal magnification of 81,000 and the pixel size was 0.88 Å/pix. The total electron dose and frame rate were 65 electrons/Ų and 0.1 s, respectively.