Vitrobot mark 4 plunger

5 μL of the complex at 3 mg/ml concentration was loaded onto a freshly glow discharged (45 s at 15 mA) holey carbon grid (Quantifoil Au R1.2/1.3) before sample application. Then, the excess liquid is removed by Whatman #1 filter paper at a blot force of 4 s at a blot force of -3 at 4°C, and 100% humidity using a Vitrobot Mark IV plunger (ThermoFisher Scientific) and plunge-frozen in liquid ethane. The prepared frozen-hydrated specimen was loaded on an FEI Titan Krios transmission electron microscope equipped with a Gatan K2 Summit direct detector and Gatan BioQuantum energy filter used for the Date collection. 5,730 movies were collected using SerialEM software that operated the 300 kV transmission electron microscope at a nominal magnification of 165,000× with a pixel size of 0.84 Å, and the movies were recorded with a defocus range of -1.2 to -1.8 μm in super-resolution mode.

Three μl of purified HKU1 HE (4 mg/ml) was dispensed on Quantifoil R1.2/1.3 200-mesh grids (Quantifoil Micro Tools GmbH) that had been freshly glow discharged for 30 seconds at 20 mA using GloQube Glow Discharge system (Quorum Technologies). Grids were blotted for five seconds using Whatman No. 1 filter paper and immediately plunge-frozen into liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV plunger (Thermo Fisher Scientific) equilibrated to ~95% relative humidity, 4 °C. Movies of frozen-hydrated HKU1 HE were collected using Titan Krios G4 Cryo-TEM (Thermo Fisher Scientific) operating at 300 keV and equipped with a Falcon 4 Direct Electron Detector (Thermo Fisher Scientific). All cryo-EM data were acquired using the EPU 2 software (Thermo Fisher Scientific). Microscope was aligned to produce fringe-free imaging (FFI) allowing five acquisition areas within a hole and aberration-free image shift (AFIS) was used to acquire images from up to 21 holes per single stage move. Movies were collected in electron counting mode at 96,000× corresponding to a calibrated pixel size of 0.805 Å/pix over a defocus range of −1.0 to −2.5 μm. 6,029 movies were collected using a dose rate of 5 e⁻/pix/s for a total of 5.6 s (207 ms per fraction, 27 fractions), resulting in a total exposure of ~40 e⁻/Å (1.5 e⁻/Ų/fraction).

Cryo-EM grids were prepared with the Vitrobot Mark IV plunger (FEI) set to 4 °C and 100% humidity. Three-microliter of the sample was applied to the glow discharged gold R1.2/1.3 holey carbon grids. The sample was incubated for 10 s on the grids before blotting for 3 s (double-sided, blot force −2) and flash-frozen in liquid ethane immediately.
For Omicron BA.2 spike trimer-hACE2 complex, Omicron BA.2 spike trimer-JMB2002 antibody complex, Omicron BA.2 spike trimer-mACE2 complex, and Omicron BA.1 spike trimer-mACE2 complex datasets, 16107, 22686, 7071, and 6545 movies were collected respectively on a Titan Krios equipped with a Gatan K3 direct electron detection device at 300 kV with a magnification of 105,000, corresponding to a pixel size 0.824 Å. Image acquisition was performed with EPU Software (FEI Eindhoven, Netherlands). We collected a total of 36 frames accumulating to a total dose of 50 e⁻Å⁻² over 2.5 s exposure.

Quantifoil Au R1.2/1.3 grids were glow-discharged at 5 mA for 1 min using an Emitech K100X Glow Discharge Unit. About 2.1 μl of sNS1 (0.25 mg/mL) / sNS1 complexed with Fab 5E3 (molar ratio 1:1.2, final concentration of sNS1 is 0.25 mg/mL) were applied to the grids, blotted with a filter paper (Ted Pella Standard Vitrobot filter paper, Grade 595) for 2 s (Blot force 2, Blot Time 2 s, drain time 0.5 s, Humidity 100%) to remove excess sample, and then flash frozen in liquid ethane by using the Vitrobot Mark IV plunger (FEI, Netherlands). To ensure temperature consistency, the temperature of the Vitrobot humidity chamber was adjusted to 4 °C for blotting.

Quantifoil R2/1 or R1.2/1.3 300 mesh holey carbon grids were glow discharged for 2 minutes at 20 mA. The freshly prepared 3 µL protein samples at a concentration of 0.5 mg/ml were added to the carbon grids and incubated for 10 seconds before plunging into liquid ethane using FEI Vitrobot Mark IV plunger at 20°C with 100% humidity. The grids were blotted for 3-5 seconds at zero blot force. Cryo-EM data acquisition of the frozen sample was carried out using a Thermo Scientific™ Talos Arctica transmission electron microscope at 200 kV at a nominal magnification of 42,200 x, equipped with a K2 Summit Direct Electron Detector. LatitudeS automatic data collection software (Gatan Inc) was utilized to collect the final 746 digital micrographs at a pixel size of 1.2 Å with a total electron dose of about 40 e-/Ų at the defocus range of −1.25 and −3.5 µm, under a calibrated dose of about 2 e-/Ų per frame. Movies were recorded for 8 secs with 20 frames. All the collected micrographs were then motion corrected using “dosefgpu_driftcorr” using MotionCor2 software (39 (link)).

Cryo-EM grids were prepared with the Vitrobot Mark IV plunger (FEI) set to 8 °C and 100% humidity. Approximately 3 μL of the purified sample was deposited onto the glow discharged Ted Pella lacey copper grids (Ted Pella, 01824; PELCO easiGlow) coated with a thin layer of continuous carbon film. A blot force of −1 and blot time of 3 s were applied to blot the grids after incubation of the sample with grids for 10 s. Then the samples on grid were vitrified by plunge freezing in pre-cooled liquid ethane at a liquid nitrogen temperature.
All micrographs were acquired on a FEI Titan Krios G3i operated at 300 keV, equipped with Gatan K3 direct electron detector. Automated image acquisition was performed with EPU Software (FEI Eindhoven, the Netherlands) at a magnification of 81,000×, corresponding to a pixel size 0.55 Å. The K3 detector was gain-corrected and micrographs were collected at a defocus varying between −1.5 μm and −2.8 μm. We collected a total of 40 frames, amounting to a total dose of 62 e^–/Ų over 3.0 s of exposure.