The cryo-EM grids were prepared using Vitrobot Mark IV (FEI) operated at 4 °C and 100% humidity. For samples of hPANX1 in digitonin, 4 μl aliquots of samples at concentrations of approximately 8 mg/ml were applied onto glow-discharged holey carbon grids (Quantifoil R1.2/1.3) 300 mesh Au grid. After a waiting time of 5 s, the grids were blotted for 2 s and plunged into liquid ethane for quick freezing.
The cryo-EM grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II 4k × 4k camera (FEI). The qualified grids were transferred into a Titan Krios microscope (FEI) operated at 300 kV for data acquisition equipped with Gatan K2 Summit detector and GIF Quantum energy filter. Images were automatically recorded using AutoEMation with a slit width of 20 eV on the energy filter and in the super-resolution mode at a nominal magnification of 130,000×, corresponding to a calibrated pixel size of 1.08 Å at the object scale, and with defocus ranging from 1.4 to 1.9 μm. Each stack was exposed for 5.6 s with an exposing time of 0.175 s per frame, resulting in a total of 32 frames per stack, and the total dose rate for each stack was about 50 e/Å2.
The cryo-EM grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II 4k × 4k camera (FEI). The qualified grids were transferred into a Titan Krios microscope (FEI) operated at 300 kV for data acquisition equipped with Gatan K2 Summit detector and GIF Quantum energy filter. Images were automatically recorded using AutoEMation with a slit width of 20 eV on the energy filter and in the super-resolution mode at a nominal magnification of 130,000×, corresponding to a calibrated pixel size of 1.08 Å at the object scale, and with defocus ranging from 1.4 to 1.9 μm. Each stack was exposed for 5.6 s with an exposing time of 0.175 s per frame, resulting in a total of 32 frames per stack, and the total dose rate for each stack was about 50 e/Å2.