Cryo-electron microscopy grid preparation and data imaging

For the Talos Arctica dataset, the purified complex was diluted to 0.16–0.2 mg/ml in 20-mM HEPES pH 7.4, 200-mM NaCl, 2-mM MgCl₂, and 0.5-mM TCEP, and incubated with BeF₃⁻ overnight. 3.5 μL of sample was deposited onto freshly glow-discharged (PELCO easiGlow, 30 s in air at 20 mA and 0.4 mbar) C-flat 1.2/1.3-3Au grids (Electron Microscopy Sciences), and blotted for 4 s using a Vitrobot Mark IV (FEI) with 30-s incubation, blot force 8 and 100% humidity and temperature set to 22 °C. The complex was visualized in the Talos Arctica operated at an acceleration voltage of 200 kV, and equipped with a Gatan K3 Summit director electron detector in super-resolution counting mode at 36,000×, corresponding to a pixel size of 0.57 Å. In total, 2579 movies were acquired with the defocus range between −1 and −2.5 μm with a 3 × 3 image shift pattern prior to stage movement. All movies consisted of 50 frames with a total dose of 50 e⁻/Ų and a total exposure time of 5.16 s.

For the untilted Titan Krios dataset, grids were prepared in the same manner and the complex was collected using a Titan Krios G2 electron microscope (FEI) operating at 300 kV with a Gatan Quantum energy filter (operated at 20-eV slit width) using a K3 summit direct electron detector (Gatan, Inc.) in super-resolution counting mode at magnification 81,000×, corresponding to a pixel size of 0.47 Å on the specimen level. In total, 10,188 movies were collected with defocus of −1.2 to −2.5 μm with a 3 × 3 image shift pattern prior to stage movement. Movies consisted of 50 frames, with a total dose of 50 e⁻/Ų and a total exposure time of 2.929 s.

The Titan Krios dataset with a stage tilt of 30° was obtained using 4-μL sample applied on freshly glow-discharged (PELCO easiGlow, 25 s in air at 25 mA and 0.4 mbar) UltrAufoil R1.2/1.3 grid (Ted Pella) and plunged into liquid ethane after 30-s incubation and blotting for 4 s using a Vitrobot Mark IV (FEI) with blot force 0 and 100% humidity and temperature set to 5 °C. Four thousand five hundred and eighty-five movies were collected using SerialEM at around −1.5–2.8-μm defocus with 1 × 3 image shift pattern. The movies were exposed to a total dose of 50 e⁻/Ų for 5.326 s at CDS mode.

The Titan Krios dataset with a stage tilt of 40° was collected using 4 μL sample applied on freshly glow-discharged (PELCO easiGlow, 25 s in air at 25 mA and 0.4 mbar) GF-1.2/1.3-3Au-45nm-50 grid (Electron Microscopy Sciences) and plunged into liquid ethane after 30-s incubation and blotting for 3 s using a Vitrobot Mark IV (FEI) with blot force 8 and 100% humidity and temperature set to 4 °C. Six thousand one hundred and seventy-eight movies were collected using SerialEM at −1.5–2.8-μm defocus with 1 × 3 image shift pattern. The movies were exposed to a total dose of 50 e⁻/Ų for 5.326 s at CDS mode.

All of the data were acquired with SerialEM using custom macros for automated single particle data acquisition. Imaging parameters for the dataset are summarized in Supplementary Table ².