300 kv titan krios

An aliquot (3.5 µl) of purified NPY–Y₁R–G_i1–scFv16 complex was applied onto a glow-discharged holey carbon grid (Quantifoil R1.2/1.3, 300 mesh). The grids were blotted for 5 s at 12 °C and 100% humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, USA) at Center for Macromolecular and Cell imaging of Seoul National University (SNU CMCI). Grids were initially screened with the FEI Glacios (Thermo Fisher Scientific, USA) at SNU CMCI, equipped with a Falcon 4 detector. Images were acquired on a 300-kV Titan Krios (Thermo Fisher Scientific, USA) at Korea Basic Science Institute, equipped with a Falcon 3EC direct electron detector. Movies were recorded in counting mode at a magnification of ×161,850 (corresponding to a calibrated pixel size of 0.865 Å) and a defocus range of −1.25 to −2.75 μm. A total of 4965 movies were collected, each comprising 40 frames, with a total dose of 40 electrons per Ų. A detailed description of the cryo-EM data collection parameters is provided in Supplementary Table 1.

Holey carbon grids (Quantifoil Au 300 mesh, R1.2/1.3) were glow-discharged in the Plasma Cleaner PDC-32G-2 (Harrick Plasma Company) with a vacuum for 2 min and mid force for 30 s. Aliquots (4 µl) of SIDT2 proteins were placed on the glow-discharged grids, which were then blotted for 3 s and flash frozen in liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific) at 8 °C and 100% humidity. The grids were loaded onto a 300 kV Titan Krios (Thermo Fisher Scientific Inc.) equipped with K3 Summit detector (Gatan) and GIF Quantum energy filter. Images were automatically collected using AutoEMation^{49 (link)} in super-resolution mode at a nominal magnification of 81,000 × (64,000 × for apoSIDT2-pH 7.4 dataset), with a slit width of 20 eV on the energy filter. A defocus series ranging from −1.3 μm to −1.8 μm was used. Each stack was exposed for 2.56 s with an exposure time of 0.08 s per frame, resulting in a total of 32 frames per stack and the total dose was approximately 50 e-/Ų for each stack. The stacks were motion corrected with MotionCor2^{50 (link)} and binned 2 fold, resulting in a pixel size of 1.0825 Å/pixel (1.0979 Å for apoSIDT2-pH 7.4 dataset). Meanwhile, dose weighting was performed^{51 (link)}. The defocus values were estimated with Gctf^{52 (link)}.

Quantifoil R1.2/1.3 Cu 300 mesh grids were glow discharged for 60 s in H₂-O₂ condition. 4 μL of Ca_V3.3^EM at ~4 mg/ml was applied to the grid followed by blotting for 5.0 s at 100% humidity and 4 °C, and flash-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, USA).
Grids were imaged with a 300 kV Titan Krios (Thermo Fisher Scientific, USA) equipped with a K2 Summit direct electron detector (Gatan, USA) and a GIF-Quantum energy filter. The slit width was set to 20 eV. A calibrated magnification of 105,000× was used, yielded a pixel size of 1.36 Å on images. The defocus range was set to between −1.2 and −2.2 μm. All movie stacks were collected using SerialEM^{51 (link)} under a dose rate of 9.1–9.4 e⁻/pixel/s with a total exposure time of 11.4 s, and dose-fractioned to 32 frames, resulting in a total dose of 60 e⁻/Å2.