Samples were prepared using human Kv1.3 protein (3 mg/mL) without a binding partner, or with nanobody at 1.3 mg/mL (3:1 molar ratio, nanobody:Kv1.3 subunit), or with Fab-ShK at 0.4 mg/mL (1:1 molar ratio, Fab-ShK:Kv1.3 tetramer). UltrAuFoil 1.2/1.3 300 mesh grids (Quantifoil) were plasma treated and vitrified samples were prepared by adding a 2.5 µL droplet of sample solution to a grid, then blotting (2 sec blot time, 0 to −4 blot force range) and plunge-freezing in liquid ethane using a Vitrobot Mk IV (Thermo Fisher).
Single particle images were collected with a Titan Krios electron microscope (Thermo Fisher) operated at 300 kV and a nominal magnification of 105,000× and equipped with a K3 camera (Gatan) set in super-resolution mode (0.4260 Å pixel size). Leginon was used for automated collection of images with ice thickness ranging between 20 nm and 120 nm71 (link),72 (link). Movies were collected at nominal defocus values of ~1.0–2.0 µm and dose-fractionation into 48 frames with a total exposure time of 2.4 sec and total dose of ~51 e-Å−2.
Single particle images were collected with a Titan Krios electron microscope (Thermo Fisher) operated at 300 kV and a nominal magnification of 105,000× and equipped with a K3 camera (Gatan) set in super-resolution mode (0.4260 Å pixel size). Leginon was used for automated collection of images with ice thickness ranging between 20 nm and 120 nm71 (link),72 (link). Movies were collected at nominal defocus values of ~1.0–2.0 µm and dose-fractionation into 48 frames with a total exposure time of 2.4 sec and total dose of ~51 e-Å−2.