K2 summit counting camera

Aliquots of 3.5 μl concentrated apo Ca_v2.2 or Ca_v2.2–ziconotide were loaded onto glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh for Ca_v2.2–ziconotide, Quantifoil Au R1.2/1.3, 300 mesh for the apo channel), which were blotted for 6 s and plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher) at 8 °C with 100% humidity. Grids were transferred to a Titan Krios electron microscope (Thermo Fisher) operating at 300 kV and equipped with a Gatan Gif Quantum energy filter (slit width 20 eV) and spherical aberration (Cs) image corrector. Micrographs were recorded using a K2 Summit counting camera (Gatan) in super-resolution mode with a nominal magnification of 105,000×, resulting in a calibrated pixel size of 0.557 Å. Each stack of 32 frames was exposed for 5.6 s, with an exposure time of 0.175 s per frame. The total dose for each stack was about 50 e⁻ per Ų. SerialEM was used for fully automated data collection^{38 (link)}. All 32 frames in each stack were aligned, summed and dose-weighted using MotionCorr2^{39 (link)} and twofold-binned to a pixel size of 1.114 Å per pixel. The defocus values were set from −1.9 to −2.1 μm and estimated by Gctf^{40 (link)}.