Pelco easiglow glow discharge unit

For cryo-EM, SARS-CoV-2 S trimer Beta mutant were deposited on grids at a final concentration of 2 mg/mL. Complexes were prepared by incubating S trimer Beta mutant with VH F6 at a molar ratio of 1:10. Grids were cleaned with H2/O2 gas mixture for 15 s in PELCO easiGlow glow discharge unit (Ted Pella) and 1.8 μL of protein suspension was applied to the surface of the grid. Using a Vitrobot Mark IV (Thermo Fisher Scientific), the sample was applied to either Quantifoil Holey Carbon R1.2/1.3 copper 300 mesh grids or UltrAuFoil Holey Gold 300 mesh grids at a chamber temperature of 10°C with a relative humidity level of 100%, and then vitrified in liquid ethane after blotting for 12 s with a blot force of −10. All cryo-EM grids were screened using a 200-kV Glacios (Thermo Fisher Scientific) TEM equipped with a Falcon4 direct electron detector and data were collection on a 300-kV Titan Krios G4 (Thermo Fisher Scientific) TEM equipped with a Falcon4 direct electron detector in electron event registration (EER) mode. Movies were collected at 155,000× magnification (physical pixel size 0.5 Å) over a defocus range of −3 μm to −0.5 μm with a total dose of 40 e – /Å2 using EPU automated acquisition software (Thermo Fisher).

Purified RNase E‐NTD, full‐length RapZ and full‐length GlmZ RNA were incubated in a ratio of 5:25:10 μM in reaction buffer (25 mM HEPES pH 7.5, 300 KCl and 1 mM MgCl₂) supplemented with 0.01% glutaraldehyde at RT for 20 min followed by at 30°C for 10 min. The cross‐linking reaction was quenched by adding 50 mM Tris pH 7.5. Samples were run on to a Superose 6 size‐exclusion column equilibrated with the reaction buffer. Fractions showing evidence for protein elution (judged by SDS–PAGE) with high 260/280 ratio were pooled, concentrated to 1 mg/ml and applied to Quantifoil Cu 300 1.2/1.3 grids coated with graphene oxide (GO) (Russo & Passmore, 2014 ; Palovcak et al, 2018 (link)). Briefly, grids were glow‐discharged on the darker carbon side in a PELCO easiGLOW glow discharge unit equipped with an oil pump (TED PELLA Inc., USA) using the following conditions: 15 mA, 0.28 mBar, 2 min. GO solution (2 mg/ml dispersion in water, Sigma‐Aldrich product code: 763705) was 10x diluted using ultrapure water (ddH₂O) and centrifuged at 300 g for 30 s to remove insoluble GO flakes. The supernatant was further diluted another 10× to make a 0.02 mg/ml GO solution. On the glow‐discharged side of the Quantifoil grids, 1 μl of 0.02 mg/ml GO solution was applied and waited until the water evaporated. GO‐coated grids were left at room temperature for at least 12–16 h before use to prepare EM specimens.

Full methods used have been published previously^{9 (link)}. Briefly samples were loaded onto carbon-coated 300 mesh copper grids (Electron microscopy Sciences) that were glow discharged for 40 seconds using an PELCO easiGLOW™ glow discharge unit (Ted Pella Inc, USA). Samples were left to bind for 2 minutes, blotted, washed briefly in one water drop, blotted, and then stained with either NanoW stain (Nanoprobes) 2 × 1 min or 2% (w/v) uranyl acetate in water for 45 sec, then blotted and air-dried. Grids were inserted into the microscope using a dedicated sample holder for mouse prions with strict adherence to risk assessment and microbiological containment level 2 safe working practice. The sample holder was decontaminated directly after use by exposing to plasma using a Fischione 1020 plasma cleaner. Images were acquired on an FEI Tecnai T10 electron microscope (FEI, Eindhoven, NL) operating at 100 kV and recorded on a 1 k × 1 k charged couple device (CCD) camera (Gatan) at a nominal magnification of 44,000 with a pixel size of 3.96 Å. Fibril dimensions were measured in ImageJ^{41 (link)} and IMOD^{42 (link)}. Because of their helical twist, prion rods and recombinant PrP fibrils viewed on a surface alternate between wider, face-on and narrower, edge-on views of the structure. The dimensions reported here were measured on the widest parts of the fibrils.