K3 direct electron detector

Cryo-EM imaging was performed on a FEI Titan Krios microscope (Thermo Fisher Scientific) operated at 300 kV. Data collection images were acquired with a Falcon 3EC or K3 Direct Electron Detector operated in counting mode with a calibrated physical pixel size of 1.08 Å with a defocus range between −1.0 and −3.5 µm using the EPU software (Thermo Fisher Scientific). No energy filter or C_s corrector was installed on the microscope. The dose rate used was ∼0.8 e^-/Ų s to ensure operation in the linear range of the detector. The total exposure time was 60 s, and intermediate frames were recorded every 2 s giving an accumulated dose of ∼42 e^-/Ų and a total of 30 frames per image.

The sample grids were loaded into a 300 keV Titan Krios TEM instrument (Thermo Fisher Scientific) equipped with a K3 direct electron detector (Gatan, CA, USA). The cryo-ET tilt series were collected by Tomography 4.0 software (Thermo Fisher Scientific) in a bi-directional tilt scheme: sample was tilted from 20 to −61 degrees, then from 23 to 59 degrees, with nine frames acquired at every 3-degree step. Among the 90 tilt series, 56 had 100 e⁻/Ų accumulative doses and 1.5 to 4 μm defocus, and the rest were acquired with 5 to 6 μm defocus and 120 e⁻/Ų accumulative doses. All frames were motion-corrected and dose-filtered with IMOD 4.11.6 alignframes function and combined to each tilt series via IMOD newstack function^{68 (link),69 (link)}. Tomogram reconstruction was performed with IMOD - ETOMO^{68 (link)}. The tomograms were binned four times (bin4, 5.558 Å/pixel) for the subsequent processing.

Purified SARS-CoV-2 S (HexaPro variant) at 0.2 mg/mL was incubated with 5-fold molar excess of Fab N3–1 in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN₃ at RT for 30 min. The sample was then deposited on plasma-cleaned UltrAuFoil 1.2/1.3 grids before being blotted for 4 sec with −3 force in a Vitrobot Mark IV and plunge-frozen into liquid ethane. Purified SARS-CoV-2 S with three RBDs covalently trapped in the down conformations (HexaPro-RBD-down variant, S383C/D985C^{45 (link)–47 (link)}) at 0.2 mg/mL, complexed with 2-fold molar excess of Fab A7V3, was deposited on plasma-cleaned UltrAuFoil 1.2/1.3 grids before being blotted for 3 sec with −4 force in a Vitrobot Mark IV and plunge-frozen into liquid ethane. For the HexaPro-N3–1 sample, 3,203 micrographs were collected from a single grid. For the HexaPro-RBD-down-A7V3 sample, 3,636 micrographs were collected from a single grid. FEI Titan Krios equipped with a K3 direct electron detector (Gatan) was used for imaging. Data were collected at a magnification of 22,500x, corresponding to a calibrated pixel size of 1.07 Å/pix. A full description of the data collection parameters can be found in Extended Data Tables 3–4.

A 5 μl aliquot of AAV9P31 was loaded onto a glow-discharged, carbon-coated copper grid (Cu 1.2/1.3+C, 300 mesh; Quantifoil) bearing an ultrathin layer of carbon and incubated for two minutes. The liquid was then sucked away by a filter and 3 μl of Car4 was loaded on the same grid, and incubated for another five minutes with 100% humidity. The grid was then blotted for 4.5 s with a blot force of 0 in 100% relative humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI, USA). Cryo-EM data were collected with a 300 kV Titan Krios electron microscope (FEI, USA) and a K3 direct electron detector (FEI, USA). A series of micrographs were collected as movies and recorded with -2.2 to -0.5 μm defocus at a calibrated magnification of 105,000×, resulting in a pixel size of 0.8433 Å per pixel. Statistics for data collection and refinement are summarized in S1 Table.

For cryo-EM, 3.5 μl of NKCC1 sample at ~ 8–10 mg/ml, supplemented with/without 500 µM bumetanide, was applied to a glow-discharged Au 1.2/1.3 holey, 300 mesh gold grid and blotted for 1.5 s at 4 °C, 90% relative humidity on a Vitrobot Mark III (FEI) before being plunge-frozen in liquid ethane cooled by liquid nitrogen. Data were collected on a Krios (FEI) operating at 300 kV equipped with the K3 direct electron detector at the University of Utah and Pacific Northwest Cryo-EM Center (PNCC). Movies were recorded using SerialEM, with a defocus range between −1.0 and −3.5 μm. Specifically, movies were recorded in super-resolution counting mode at a physical pixel size of 1.06 Å. The data were collected at a dose rate of 1.175 e − /Ų/frame with a total exposure of 40 frames, giving a total dose of 47 e − /Å2.