Plasma cleaner pdc 002 ce

A 3 μL aliquot of B.1.135 S ectodomain at a concentration of ∼1.2 μm with fab (1:6 molar ratio) was prepared, aspirated and almost immediately applied to a freshly glow-discharged C-flat 200 mesh 2/1 grids at high intensity, 20 s, Plasma Cleaner PDC-002-CE, Harrick Plasma. Excess liquid was removed by blotting for 5 s with a force of −1 using vitrobot filter paper (grade 595, Ted Pella Inc.) at 4.5°C, 100 % reported humidity before plunge freezing into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Fab/Spike complexes were incubated for 5–10 minutes prior to application to grids and plunge freezing.

Purified spike protein was buffer exchanged into 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN₃ buffer using a desalting column (Zeba, Thermo Fisher). A final concentration of 0.2 mg/mL was incubated with CR3022 Fab (in the same buffer) in a 6:1 molar ratio (Fab to trimeric spike) at room temperature. Aliquots were taken at 50 min and 3 h and 3 μL immediately applied to a holey carbon-coated 200 mesh copper grid (C-Flat, CF-2/1, Protochips) that had been freshly glow discharged on high for 20 s (Plasma Cleaner PDC-002-CE, Harrick Plasma) and excess liquid removed by blotting for 6 s with a blotting force of −1 using vitrobot filter paper (grade 595, Ted Pella Inc.) at 4.5°C, 100% relative humidity. Blotted grids were then immediately plunge frozen using a Vitrobot Mark IV (Thermo Fisher).
Frozen grids were first screened on a Glacios microscope operating at 200 kV (Thermo Fisher) before imaging on a Titan Krios G2 (Thermo Fisher) at 300 kV. Movies (40 frames each) were collected in compressed tiff format on a K3 detector (Gatan) in super resolution counting mode using a custom EPU version 2.5 (Thermo Fisher) with a defocus range of 0.8-2.6 μm and at a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel, see Table S4.

Preheated (30 min at 200 °C) 4 in. silicon wafer was spin‐coated (model no. WS‐650MZ‐23NPPB, Laurell Tech. Corp) as per the specifications provided by the manufacturer at 23 °C. Pre‐ and post‐baking steps are performed at 65 °C for 3 min and 95 °C for 9 min, respectively. After 8 s of UV exposure (using Kloe photolithographic instrument (model no. UV‐KUB 2) through film mask design), the wafer was post‐baked at 65 °C for 2 min and 95 °C for 7 min. Followed by a 2 min washing step with the developer solution and later with isopropanol, the wafer was baked at 200 °C for 2 h before performing overnight surface passivation with 50 µL of 1H,1H,2H,2H‐perfluoro‐decyl trichlorosilane in a desiccator. PDMS:curing agent (10:1) mixture was thoroughly mixed and degassed for 30 min at 150 millibars low pressure. The degassed mixture was poured on top of the surface‐passivated wafer and cured at 90 °C for 3 h. Thus, crosslinked PDMS was pealed from the master mold and diced into small pieces under a clean hood. A 1 mm biopsy puncher ((Kai Europe GmbH) was used to create the inlets and outlets. Finally, plasma cleaned (at 600 mbar for 1 min) (Plasma Cleaner PDC‐002‐CE, Harrick Plasma) glass coverslips and diced PDMS chips were bonded to form the microfluidic chip. These chips were further heated at 60 °C for 2 h to complete the bonding process and retention of the hydrophobic surface.