K3 camera

The samples were diluted to 20 μM in the SEC buffer supplemented with 0.05% Nonidet P-40 before cryo-EM sample preparation. Using a Vitrobot Mark IV (Thermo Fisher Scientific), 3.5 μL sample solution at 20 μM was applied to a Quantifoil 2/1 holey carbon grid at 10 °C and 95% relative humidity and vitrified by plunge freezing after removing excess liquid by blotting for 2 s with a blotting force of 1. The grids were imaged using a Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a K3 camera (Gatan) using the Serial-EM automation software (56 (link)). The nominal magnification was 130,000× and the pixel size was 0.3265 Å. The dose rate was around 20 electrons per physical pixel per second. At each stage position, a group of nine holes was imaged using the multiple record setup (56 (link), 57 ), and each hole contained six imaging spots. At each imaging spot, a 40-frame movie stack was collected with a total exposure time of 1.012 s. The data collection speed was about 9,000 movie stacks per day. More details for data collection are summarized in SI Appendix, Table S2.

Movies were collected on a 300-kV Titan Krios microscope with a GIF energy filter and Gatan K3 camera. Super-resolution pixel size was 0.355 Å, for a physical pixel size of 0.71 Å. SerialEM (Schorb et al., 2019 (link)) was used to correct astigmatism, perform coma-free alignment, and automate data collection. Movies were collected with the defocus range −0.6 to −1.5 µm and the total dose was 39.89 e^-/Ų split over 40 frames. One movie was collected for each hole, with image shift used to collect a series of 3 × 3 holes for faster data collection (Cheng et al., 2018 (link)), and stage shift used to move to the center hole. Based on the 1.2/1.3 grid hole specification, this should correspond to a maximum image shift of ~1.8 μm, although the true image shift used was not measured. The beam size was chosen such that its diameter was slightly larger than that of the hole, that is, >1.2 μm, although we have observed variation in the actual hole size compared to the manufacturer specifications.

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 nm^{71 (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.

A sample of 0.5 μM (final concentration) purified SMG1-SMG8-SMG9 was mixed with 0.5 mM of UPF1-LSQ, 1 mM AMPPNP, 2 mM MgCl₂, 2 mM DTT and 0.04% (v/v) n-octyl-beta-D-glucoside in 1xPBS and incubated for 30 min on ice. The UPF1-LSQ peptide (sequence: QPELSQDSYLG) was synthesized in-house as described for the mass spectrometry-based phosphorylation assay. A 4 μL sample was applied to a glow-discharged Quantifoil R1.2/1.3, Cu 200 mesh grid and incubated for 30 s at 4°C and approximately 100% humidity. Grids were subsequently plunge frozen directly after blotting using a liquid ethane/propane (37% ethane, temperature range when plunging: −170°C to −180°C) mixture and a ThermoFisher FEI Vitrobot IV set to a blot time of 3.5 s and a blot force of 4. Cryo-EM data were collected using a ThermoFisher FEI Titan Krios microscope operated at 300 kV equipped with a post-column GIF (energy width 20 eV) and a Gatan K3 camera operated in counting mode, the SerialEM software suite, and a beam-tilt based multi-shot acquisition scheme. Movies were recorded at a nominal magnification of 81.000x corresponding to a pixel size of 1.094 Å at the specimen level. The sample was imaged with a total exposure of 68.75 e^-/Ų evenly spread over 5.5 s and 79 frames. The target defocus during data collection ranged between −0.8 and −2.9 μm.

A screening dataset was collected manually on an F20 instrument (FEI) using the UCSF Figure 4 semi-automated data collection software package (Li et al., 2015 (link)). A single large dataset was collected on a Titan Krios G3i (FEI). The instrument was outfitted with a pre-camera energy filter (Gatan Image Filter) and operated at 300 kV. Images were collected in nanoprobe mode at spot size three with an illuminated area of ~1 μm at the sample level. Images were collected on a K3 camera (Gatan) in counting mode at a nominal magnification of 105 kx with a pixel size of 0.85 Ų (Table 4). Dose-fractionated movies were collected (3 s per movie, 50 frames, 17 electrons per Ų per second, and 1.0 electrons per Ų per frame). Five movies were collected for each hole on the grid, and nine holes were imaged at each stage position, yielding 45 movies per round of stage movement. We used SerialEM v3.7 with on-the-fly beam tilt correction of image shift-induced coma to collect the high-resolution dataset (Mastronarde, 2005 (link)). Computational beam tilt correction during data processing further improved the experimental density (Figure 1—figure supplement 1).