Gloqube

Sample volumes of 3.5 µl (8 OD_260 nm per ml) were applied to grids (Quantifoil, Cu, 300 mesh, R3/3 with 3 nm carbon) which had been freshly glow-discharged using a GloQube (Quorum Technologies) in negative charge mode at 25 mA for 90 s. Sample vitrification was performed using ethane or propane in a Vitrobot Mark IV (Thermo Fisher Scientific), the chamber was set to 4 °C and 100% relative humidity and blotting was done for 3 s with no drain or wait time. Data were collected in an automated manner using EPU v.3.0 on a cold-FEG fringe-free Titan Krios G4 (Thermo Fisher Scientific) transmission electron microscope operating at 300 kV. The camera was operated in electron counting mode and data were collected at a magnification of 96,000× with the nominal pixel size of 0.83 Å and a nominal defocus range of −0.4 to −0.9 μm. A total of 23,349 micrographs in EER format were collected with 5.31 s of exposure (corresponding to a total dose of 50 e per A² on the specimen). No statistical methods were used to predetermine the sample size. The sample size was selected on the basis of a three-day data collection, which was chosen to obtain a sufficient number of particles for data processing.

Specimens were prepared by
plunge freezing suspensions (at the original concentration) on copper
grids (300 mesh) containing lacey carbon film. Prior to use, the grids
were glow discharged using a Quorum Technologies GloQube instrument
at a current of 25 mA for 60 sec. The sample (3 μL) was pipetted
onto a TEM grid, blotted for 3 sec at blot force -5 using dedicated
filter paper, and immediately plunged into liquid ethane using a Vitrobot
Mark IV. The Vitrobot chamber was set to 4 °C and 95% humidity.
Specimens after vitrification were kept under liquid nitrogen until
they were inserted into a Gatan Elsa cryo holder and imaged in the
TEM at −178 °C. Images were collected using a Thermo Scientific
(FEI) Talos F200X G2 microscope at 200 kV at low dose using a Ceta
16M CMOS camera.

Quantifoil R2/1 holey carbon grids (Au 300 mesh, Electron Microscopy Sciences) were glow-discharged for 60 s at 20 mA using a GloQube (Quorum) instrument. Purified Nf1-23a was thawed, centrifuged (14,000g for 5 min at 4 ºC) and diluted to ~0.5 mg ml⁻¹ with gel filtration buffer. Protein was loaded into the freshly glow-discharged grids and plunge-frozen in LN₂-cooled liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) with a blot force of −2 for 2.5 s. Temperature and relative humidity were maintained at 4 ºC and 100%, respectively. Grids were clipped and loaded into a 300-kV Titan Krios G2 microscope (Thermo Fisher Scientific, EPU 2.8.1 software) equipped with a Gatan BioQuantum energy filter and a K3 Summit direct electron detector (AMETEK). Grids were screened for quality control based on particle distribution and density, and images from the best grid were recorded. Micrographs were recorded at a nominal magnification of ×105,000, corresponding to a calibrated pixel size of 0.86 Å. The dose rate was 12 electron physical pixels per second, and images were recorded for 3.3 s divided into 40 frames, corresponding to a total dose of 40 electrons per Ų. Defocus range was set between −0.5 μm and −4 μm. Gain-corrected image data were acquired.

Two samples at different concentrations (10 and 40 µM) of NiV and HeV W were prepared and analyzed at different times to monitor their evolution. Incubation was carried out at 37 °C in HBS pH 7. Prior to each measurement, the samples were diluted to reach a final concentration of 1.6 µM. Drops of 3 μL of the diluted solution were deposited onto a glow discharge carbon coated grid (Carbon 300 mesh 3 mm Cu, TAAB). Prior to protein deposition, grids were exposed to plasma glow discharge for 20 s using a GloQube (Quorum, Lewes, UK) (Current 25 mA) in order to increase protein adhesion. The grids were washed three times with 50 µL of buffer, then washed in 35 µL 2% (w/v) Uranyl Acetate solution (LauryLab, Brindas, France) before incubating them for 1 min in the latter solution. Excess of uranyl was blotted and grids were dried for 1 h at RT. Images were collected using a TECNAI T12 Spirit microscope (Thermo Fisher Scientific, Waltham, MA, USA) operated at 120 kV, and a Veleta 2 K × 2 K CCD camera (Olympus, Shinjuku, Tokyo, Japan).

All the variants, at a concentration of 200 µM, were prepared and analyzed at different times to monitor their evolution (0, 24, 96 h). Incubation was carried out at 37 °C in Buffer C. Prior to each measurement, the samples were diluted to reach a final concentration of 40 µM. EM grids (carbon coated copper grids, 300 mesh, Agar Scientific, UK) were exposed to plasma glow discharge for 20 s using GloQube (Quorum, UK) (Current 15 mA) in order to increase protein adhesion. Drops of 3.5 μL of the diluted protein solutions were deposited onto glow-discharged grids. After 1 min incubation with the sample, the grids were washed three times with 50 µL of buffer C, once in 35 µL 1% (w/v) Uranyl acetate solution (Laurylab, Brindas, France) and then stained for 1 min in the latter solution. Excess of uranyl was blotted and grids were left to dry for 1 h at RT. Images were collected on Tecnai 120 Spirit TEM microscope (FEI company, ThermoFisher, Illkirch-Graffenstaden France) operated at 120 kV using a Veleta 2K × 2K CCD camera (Olympus).