Talos f200c transmission electron microscope

A volume of 5 μL of isolated EVs was transferred to formvar-carbon-coated copper electron microscopy grids following the protocol of Thery et al. [91 (link)], with modifications. First, 5 μL of sample were mixed with 5 μL of paraformaldehyde (4%). The sample was transferred to the grid for 20 min and washed on a PBS drop. The samples were fixed by putting the grid on a glutaraldehyde (1.5%) drop for 5 min, washed several times in distilled water drop, and transferred to 0.5% of uranyl oxalate (Electron Microscopy Sciences, Hatfield, PN, USA) (pH 7.0) drop for 5 min to contrast the grid. Then, the grid was transferred to a 4% uranyl acetate drop for 10 min and left to dry on filter paper. Finally, each grid was placed on the TEM sample support to perform the search and acquisition of EV images at a magnification of 40,000× to 80,000× in the Talos™ F200C transmission electron microscope (Thermo Fisher Scientific). Approximately 5 images per sample were captured and processed with ImageJ software (V1.47t, NIH, Bethesda, MD, USA), the link to which is available at electronic address https://imagej.nih.gov/ij/download.html, accessed on 25 December 2021.

C. crescentus ΔrogA cells (in 200-mL culture) were grown in PYE at 28 °C to stationary phase. Cells were pelleted by centrifugation (8,000 rpm for 10 min at 4 °C). The supernatant that contained GTA particles was then transferred to a fresh 500-mL bottle and was filtered twice using a 0.22-μm filter funnel (Sartorius). The supernatant was concentrated using a 100-kDa MWCO Amicon concentrator (Merck) to approximately 25 mL. GTA particles in this concentrated supernatant were precipitated by incubating with a 5xPEG/NaCl solution [20% PEG-8000 and 2.5 M NaCl] on ice for 30 min. Precipitated GTA particles were collected by centrifugation (8,000 rpm for 20 min at 4^°C) and were resuspended in 200 μL storage buffer [50 mM Tris pH 8.0, 150 mM NaCl, and 5% glycerol].
For TEM analysis, 3 μL of the purified GTA particles was pipetted on a 400-mesh copper grid (EM Resolutions) that had been glow discharged for 20 s at 10 mA in an Ace 200 (Leica Microsystems). After 60 s, excess solution was wicked away using Whatman No. 1 filter paper and leftover samples on the grid were stained using 2% (w/v) uranyl acetate solution. Grids were imaged using a Talos F200C transmission electron microscope (Thermo Fisher Scientific) operated at 200 kV, equipped with a 4 k OneView CMOS detector (Gatan, UK).

X-bodies or square-bodies were diluted to 200 nM in 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl. A 3.5 µL aliquot of diluted antibody was applied to the formvar/carbon film of a glow discharged 300 mesh copper grid (EMS, cat # FCF300-Cu-50). After removing the excess sample, the film was negatively stained with 1% uranyl acetate and dried in the air. Micrographs were collected using a Talos F200C transmission electron microscope (Thermo Fisher Scientific) equipped with a Ceta CCD camera, with a pixel size of 1.4 Å. Two hundred images were recorded using EPU software (Thermo Fisher Scientific) with a defocus range of -1.5~-2.5 μm. CTF estimation was performed using CTFFIND4.

FtsZ filament morphology was visualized by negative staining and TEM. For FtsZ from S. venezuelae, 3.5 µM FtsZ and/or 0.6 µM SepH was prepared in buffer P (50 mM HEPES pH 7.2, 50 mM KCl, 5 mM MgCl₂). All the solutions were previously filtered using 0.1 µm centrifugal filter units (Millipore). Reactions were pre-warmed at 30°C for 10 min, started by adding 2 mM GTP, and incubated at 30°C for an additional 15 min. 1 or 3.5 μL of each reaction was placed on a carbon-filmed, 400 mesh copper grid (EM Resolutions, Sheffield, UK) which had been glow discharged for 20 s at 10 mA in an Ace 200 (Leica Microsystems (UK) Ltd, Milton Keynes, UK). After 60 s, excess sample was wicked away using Whatman No. 1 filter paper and grids were negatively stained using 2% (w/v) uranyl acetate in water. Grids were imaged using a Talos F200C transmission electron microscope (ThermoFisher Scientific, Eindhoven, The Netherlands) operated at 200 kV, equipped with a 4 k OneView CMOS detector (Gatan UK, Abingdon, Oxfordshire, UK).
For M. smegmatis proteins, 6 µM FtsZ_Ms was prepared in modified buffer P (50 mM HEPES pH 6.8, 100 mM KCl, 5 mM MgCl₂) in the absence or presence of SepH_Ms at 3 µM or 6 µM. Reactions were pre-warmed to 37°C for 10 min, and then started by adding 2 mM GTP and incubated for further 20 min. Samples were stained and imaged as described above.

Purified Ab 2-7 scFv was mixed with purified SARS-CoV-2 S D614G (ExcellGene SA) at a molar ratio of 1.5:1 (Ab 2-7 to spike). Spike protein was purified by size exclusion prior to complex formation to remove soluble aggregates. The sample was incubated at room temperature for 30 min, deposited onto gold cryo-EM grids (UltrAuFoil 1.2/1.3), which had been plasma cleaned for 3 min using a Solarus 950 plasma cleaner (Gatan) with a 4:1 ratio of O2/H2. The excess protein sample was blotted away (Vitrobot filter paper; -3 force, 4 s blot time, 100% humidity, room temperature) before plunge freezing in liquid ethane (Vitrobot, ThermoFisher). Grids were screened for quality on a Talos F200C transmission electron microscope equipped with a Ceta 16 M detector (ThermoFisher). Grids that passed quality control were loaded onto a Titan Krios (ThermoFisher) operating at 300 kV, equipped with a K3 Detector (Gatan). The pixel size was 0.66 Å. Motion correction and CTF estimation were performed in WARP. Particle picking, 2D classification, 3D reconstruction and refinement were performed in cryoSPARC. Model building was performed iteratively using Coot, Isolde, and Phenix. A detailed description of image collection, processing and model quality is shown in Supplementary Figs. 2, 3.