K2 summit

Before making cryo-grids, the heparin-induced assembly reactions were centrifuged at 100,000 g for 30 min at 4°C. The resulting pellets were resuspended in 20 mM Tris, pH 7.4, 100 mM NaCl. Pronase-treated tau filaments (3 μl, at 2.0 mg/ml) were applied to glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh), blotted with filter paper and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV. For 2N4R filaments, imaging was performed on an FEI Tecnai G2 Polara microscope operating at 300 kV using a Falcon III detector prototype in integrating mode. A total of 717 movies of 30 frames was recorded during 1.0 s exposures, at a pixel size of 1.38 Å on the specimen, and a total dose of approximately 48 e/Ų. Defocus values ranged from −1.7 to −2.8 μm. For 2N3R filaments, imaging was performed on a Gatan K2-Summit detector in counting mode, using an FEI Titan Krios at 300 kV. A GIF-quantum energy filter (Gatan) was used with a slit width of 20 eV to remove inelastically scattered electrons. A total of 2051 movies of 44 frames was recorded during 11 s exposures, at a pixel size of 1.04 Å on the specimen, and a total dose of 50 electrons per Ų. Defocus values ranged from −0.8 to −2.2 μm. Further details are presented in Table 1.

PvHK at a concentration of 5 mg ml⁻¹ in buffer [20 mM Tris pH 7.5, 50 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP)] was applied to Quantifoil Cu 200 mesh grids (1.2/1.3) that were plasma cleaned using a Solarus plasma cleaner (Gatan). The sample was blotted for 3 s and plunge-frozen in liquid ethane cooled by liquid nitrogen using an FEI Vitrobot plunge-freezing instrument. The blotting chamber was maintained at 20°C and a humidity of 100%. Cryo-EM imaging was carried out with an FEI Titan Krios operating at 300 kV. Images were acquired with a K2 Summit camera placed at the end of a Gatan Imaging Filter (GIF) in super-resolution mode with a magnified pixel size of 0.4177 Å. Images were collected spanning a defocus range of 0.5–3 µm. The dose rate was ∼1.8 e⁻ per pixel per second and the exposure time was 23.2 s. Movies were collected at a rate of 2.5 frames per second, giving 58 frames per image.

For grids preparation, 3 μL concentrated protein was loaded onto glow-discharged R1.2/1.3 Quantifoil grids at 4 °C under 100% humidity. Grids were blotted for 4.5 s and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI). Micrographs were acquired on a Titan Krios microscope (FEI) operating at a voltage of 300 kV. During one collection session (KCNQ2-CaM_HN37, Supplementary Fig. 6) micrographs were collected at a magnification of 49,310× (calibrated pixel size 1.014 Å, defocus range −1.1 μm to −1.3  μm, 40 frames, and a total dose 64 e⁻/Ų) using a Gatan K2 Summit direct electron detection camera via SerialEM^{48 (link)} following standard procedure. After the microscope was upgraded with a Gatan Falcon 4 camera equipped with energy filter (Thermo Fisher Scientific) with slit width set to 10 eV, additional collection sessions (Supplementary Figs. 1, 2, 8, 9) were conducted at a magnification of 130,000× (calibrated pixel size 0.93 Å, defocus range −0.8 μm to −1.5 μm, 40 frames, and a total dose 52 e⁻/Ų) using EPU software (Thermo Fisher Scientific).