The sections (250 nm) were acquired using a Leica EM UC7 ultramicrotome (Leica) and collected on Formvar-coated copper slot grids, as previously described (Sun et al., 2021 (link)). After that, staining was performed with 2% uranyl acetate in 70% methanol and then with 0.4% lead citrate (17900; EMS). Commercial 15-nm gold particles (EMGC15; BBI Solutions) were added to both sides of the sections as fiducial markers. Dual tilt-axis series ranging from −60° to +60° were collected using an FEI Tecnai F20 electron microscope (Thermo Fisher Scientific), which was equipped with a Gatan US4000 (895) CCD camera and controlled with the FEI Xplore 3D TEM tomography software.
Fei tecnai f20 electron microscope
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
The FEI Tecnai F20 is a transmission electron microscope (TEM) that enables high-resolution imaging and analysis of samples at the nanoscale. It features a field emission gun (FEG) and advanced optics to produce a high-intensity electron beam, allowing for detailed observation and characterization of a wide range of materials, from biological specimens to advanced materials.
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Lab products found in correlation
K2 summit direct detection camera, by Ametek (2 mentions)
Em uc7 ultramicrotome, by Leica (1 mentions)
Emgc15, by BBI Solutions (1 mentions)
Glow discharged carbon coated copper grids, by Agar Scientific (1 mentions)
Field emission gun, by Thermo Fisher Scientific (1 mentions)
Vitrobot mark 3, by Thermo Fisher Scientific (1 mentions)
4 protocols using fei tecnai f20 electron microscope
1
Electron Tomography Sample Preparation
2
Negative-stain EM Analysis of Bcs Variant
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Negative-stain single-particle EM was used for direct visualization of the IMAC elution fraction of a purified BcsHisHFL variant carrying the cis-filamentation–disruptive L339D-L343D double mutation upon coexpression with BcsD (BcsHisHFL_L339D-L343D−BcsD). Briefly, 5 μl of IMAC-eluted sample (concentrations, ~0.01 to 0.02 mg/ml) was spotted on glow-discharged carbon-coated copper grids (Agar Scientific). After 60-second incubation, the extra liquid was blotted off, and the grids were passed sequentially through three drops of 2% (w/v) uranyl acetate solution, with 30-second incubation in the last drop before blotting and air drying. Micrographs were taken on a Thermo Fisher Scientific FEI Tecnai F20 electron microscope operated at an accelerating voltage of 200 kV and equipped with a field emission gun and an Eagle 4k × 4k charge-coupled device (CCD) camera. Micrographs were collected with a nominal defocus range of −1.5 to −3 μm and a low dose of ~30 electrons/Å at a pixel size of 1.835 Å2. Micrograph contrast transfer function (CTF) correction was performed with Gctf (47 (link)) through the cryoSPARC v3 interface (48 (link)), and autopicking and class averaging were performed with cryoSPARC’s “Blob picker” and 2D classification tools, respectively.
3
Cryo-EM imaging of TcdB-DLD-4 complex
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The complex of TcdB and DLD-4 was imaged under both the FEI Tecnai F20 electron microscope (FEI Company, the Netherlands) and a JEOL JEM3200FSC electron microscope (JEOL, Japan), with their respective field emission guns operated at 200 kV and 300 kV. Each microscope is equipped with a Gatan K2 summit direct detection camera (Gatan, Pleasanton, CA); 2,255 (from Tecnai F20) and 1,647 (from JEM3200FSC) micrographs were collected using macro mode or manual mode of SerialEM [70 (link)] in the super-resolution electron-counting mode. Nominal magnifications of 25,000X (on Tecnai F20) and 30,000X (on JEM3200FSC) were used, yielding subpixel sizes of 0.75 Å and 0.615 Å, respectively. The beam intensity was adjusted to 5 e−/Å2/s on the camera. A 33-frame movie stack was collected for each picture, with 0.2 seconds per frame, for a total exposure time of 6.6 seconds. For data collected on the JEM3200FSC, an in-column energy filter was used with a slit width of 29 eV.
As a control, the apo-state TcdB was imaged similarly but under the FEI Tecnai F20 electron microscope only. Image data were collected under nominal magnifications of both 25,000X (200 micrographs) and 29,000X (200 micrographs), yielding subpixel sizes of 0.75 Å and 0.62 Å, respectively.
As a control, the apo-state TcdB was imaged similarly but under the FEI Tecnai F20 electron microscope only. Image data were collected under nominal magnifications of both 25,000X (200 micrographs) and 29,000X (200 micrographs), yielding subpixel sizes of 0.75 Å and 0.62 Å, respectively.
4
Cryo-EM Structural Analysis of GroEL-PepQ
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PepQ was denatured in 8 M urea, 25 mM glycine phosphate, at pH 2 and incubated for 30 min at room temperature. Denatured PepQ (50 μM), in droplets of 4.6 μl (2.3 μM per addition) was titrated into solutions of either GroEL or Δ526 (8 μM tetradecamers, 100 μl) in TKM buffer, followed by rapid, repeated mixing and then incubated at room temperature for 5 min. The final concentration of PepQ was 7 μM. 3 μl of this PepQ/GroEL mixture was applied to a C-Flat 1.2/1.3 400 mesh holey carbon grid at 20 °C with 100% relative humidity and vitrified using a Vitrobot (Mark III, FEI company, Netherlands). The thin-ice areas that showed clear and mono-dispersed particles were imaged under an FEI Tecnai F20 electron microscope with a field emission gun (FEI company, Netherlands) operated at 200 KV. Data were collected on a Gatan K2 Summit direct detection camera (Gatan, Pleasanton CA) in electron counting mode70 (link) at a nominal magnification of × 19,000, yielding a pixel size of 1.85 Å. The dose rate was 10 e− pixel−2 s−1 at the camera. A 33-frame movie stack was recorded for each micrograph, for a total exposure time of 6.6 s. The total dose onto the specimen was 19 e− Å−2.
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