Samples were diluted to final concentrations of 20–50 μg/mL (20 mM HEPES, pH 8.0, 40 mM KOAc, 5 mM MgCl2, 0.1% trehalose, 2 mM DTT, 0.01% NP-40) and 3 μL of aliquots were applied to freshly glow discharged Quantifoil R2/1 grids coated with a second layer of thin carbon film. The grids were blotted for 3–4 s at 4 °C in 100% humility, and then plunged into liquid ethane using a FEI Vitrobot (FEI Company). Frozen grids were stored in liquid nitrogen. The grids were firstly loaded into a Gatan 626 cryo-holder and transferred to an FEI Tecnai TF20 electron microscope to check the quality of the sample vitrification. Then, the grids were transferred to Titan Krios equipped with a field emission source and operated at 300 kV. Images were recorded on a K2 direct electron detector at a nominal magnification of 18,000× with a defocus range of −2 μm to −3 μm, resulting in a calibrated sampling of 1.35 Å per pixel. The total dose was set to be 50 electrons per Å2 on the specimen and the exposure time was 11.55 s. Each image was fractionated into 50 frames.
Fei vitrobot
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FEI Vitrobot is a specialized instrument used for preparing cryo-electron microscopy (cryo-EM) samples. It is designed to rapidly freeze biological samples in a thin layer of vitreous ice, which is essential for high-resolution cryo-EM imaging. The Vitrobot precisely controls the blotting force, blotting time, and environmental conditions during the sample preparation process to ensure reproducible and high-quality sample vitrification.
Automatically generated - may contain errors
Lab products found in correlation
Titan krios, by Thermo Fisher Scientific (7 mentions)
Tecnai tf20 electron microscope, by Thermo Fisher Scientific (3 mentions)
626 cryo holder, by Thermo Fisher Scientific (2 mentions)
Ultrascan ccd camera, by Ametek (2 mentions)
Titan krios tem, by Thermo Fisher Scientific (2 mentions)
Tecnai g2 polara electron microscope, by Ametek (2 mentions)
Fei titan krios, by Thermo Fisher Scientific (2 mentions)
2 1 grid, by Quantifoil (2 mentions)
Falcon 2 direct electron detector, by BD (1 mentions)
Fibronectin, by Merck Group (1 mentions)
R2 2 finder em grids, by Quantifoil (1 mentions)
Glass bottom culture dishes, by MatTek (1 mentions)
H 7000fa, by Hitachi (1 mentions)
K2 summit direct electron detector, by Ametek (1 mentions)
Gatan quantum ls imaging energy filter, by Ametek (1 mentions)
Jem 2100, by JEOL (1 mentions)
Filter paper, by Cytiva (1 mentions)
R 2 2 cu 200 mesh, by Quantifoil (1 mentions)
K2 summit electron detector, by Ametek (1 mentions)
Krios microscope, by Ametek (1 mentions)
29 protocols using fei vitrobot
1
Cryo-EM Sample Preparation Protocol
2
Avoiding AAV-DJ Aggregation on EM Grids
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AAV-DJ was complexed with ligand on the EM grid to avoid aggregation. Aggregation was observable when mixed in bulk solution, presumably because of oligosaccharide cross-linking polyvalent AAV particles. AAV-DJ (3 μL) was applied to copper grids that had been glow discharged in 75%/25% Ar/O at a concentration of 0.6 mg/mL. For complex, the grid was hand blotted before adding 3 μL 5.7 mM fondaparinux in ultrapure water for 15 s, giving a >600-fold excess of ligand over virus subunits. All grids were vitrified in liquid nitrogen-cooled ethane using an FEI Vitrobot (FEI) with a single 3 s blot of force 1 at 100% humidity and 4°C. Grids were stored in liquid nitrogen until used.
3
Cryo-EM of NuA4 Histone Acetyltransferase Complex
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The NuA4 complex was diluted to final concentration 20–50 μg/ml (20 mM HEPES [pH 8.0], 40 mM KOAc, 5 mM MgCl2, 0.1% trehalose, 2 mM DTT, 0.01% NP-40) and 3 μl of aliquots were applied to freshly glow discharged Quantifoil R2/1 grids coated with a second layer of thin carbon film. The grids were blotted for 3–4 s at 4 °C in 100% humility, then plunged into liquid ethane using an FEI Vitrobot (FEI Company). Frozen grids were stored in liquid nitrogen. A batch of grids were prepared under the same condition and one or two grids were used to check the quality of the sample vitrification on FEI Tecnai TF20 electron microscope. The grid would be recovered if the vitrification looked good. Then, the recovered grid along with the same batch of grids would be loaded into a Titan Krios microscope for data collection. High resolution images were recorded on Titan Krios equipped with a field emission source operated at 300 kV and K2 direct electron detector at a nominal magnification of 18,000 with a defocus range of 2.0–3.0 μm, resulting in a calibrated sampling of 1.3 Å per pixel. The total accumulated dose rate was set to be 30 e2 per Å2 on the specimen and the exposure time was 7.5 s. Each image was fractionated into 30 frames.
4
Cryo-EM Sample Preparation Workflow
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Samples were diluted to a final concentration of 20–50 μg ml−1 (20 mM HEPES (pH 8.0), 40 mM KOAc, 5 mM MgCl2, 0.1% trehalose, 2 mM DTT and 0.01% NP-40), and 3 μl of aliquots was applied to freshly glow-discharged Quantifoil R2/1 grids coated with a second layer of thin carbon film. The grids were blotted for 3–4 s at 4 °C in 100% humility, and then plunged into liquid ethane using an FEI Vitrobot (FEI Company). Frozen grids were stored in liquid nitrogen. The grids were first loaded into a Gatan 626 cryo-holder and transferred to an FEI Tecnai TF20 electron microscope to check the quality of the sample vitrification. Then, the grids were transferred to Titan Krios equipped with a field emission source and were operated at 300 kV. Images were recorded on a Falcon 2 direct electron detector at a nominal magnification of × 59,000 with a defocus range of 2–4 μm, resulting in a calibrated sampling of 1.42 Å per pixel. The total accumulated dose rate was set to be 35 e2 per Å2 on the specimen, and the exposure time was 1.5 s. Each image was fractionated into 23 frames.
5
Vitrification and Cryo-EM Imaging of INO80 Complex
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The INO80 fraction was diluted to a final concentration of ~30 μg/ml (20 mM HEPES, pH 8.0, 40 mM KOAc, 5 mM MgCl2, 0.1% trehalose, 2 mM DTT, and 0.01% NP-40) and 3 μl aliquots were applied to freshly glow-discharged Quantifoil R2/1 grids coated with a second layer of thin carbon film. The grids were blotted for 3–4 sec at 4°C in 100% humility, and then plunged into liquid ethane using an FEI Vitrobot (FEI Company). Frozen grids were stored in liquid nitrogen. The grids were first loaded into a Gatan 626 cryo-holder and transferred to an FEI Tecnai TF20 electron microscope to check the quality of the sample vitrification. Then, the grids were transferred to Titan Krios equipped with a field emission source and were operated at 300 kV. Images were recorded on a Falcon II direct electron detector at a nominal magnification of 59000 with a defocus range of 3–5 μm, resulting in a calibrated sampling of 1.42 Å per pixel. The total accumulated dose rate was set to be 35 e2 per Å2 on the specimen, and the exposure time was 1.5 sec. Each image was fractionated into 23 frames.
6
Cryo-EM Structural Analysis of TAX-4 Protein
7
Cryo-EM Imaging and Tomography
8
Cryo-EM Imaging and Tomography
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Specimens were plunge-frozen onto a Quantifoil R2/2, Cu/Rh holey carbon grid using an FEI Vitrobot (FEI Company, USA) and imaged with an FEI Tecnai G2 Polara electron microscope operating at 300 kV and equipped with a Gatan energy filter and 4 × 4 k Gatan Ultrascan CCD camera (binned to 2 × 2 k). For tomography, samples were imaged using an FEI Titan Krios TEM operating at 300 kV, equipped with a 4k × 4k K2 Summit direct electron detector at a magnification of 26 k, corresponding to a pixel size of 0.45 nm (for K2) at the specimen level. Tilt series were acquired using SerialEM software 58 (link) between ± 55° with 1° increment. The defocus was set to between 5 and 8 μm, and the total dose for each tilt series was around 120 e/Å2. Tilt series was aligned using the fiducial markers and IMOD software package and reconstruction was computed using simultaneous iterative reconstruction technique (SIRT) 59 (link),60 (link).
9
Plunge-freezing of adherent cells
10
TEM and Cryo-EM Imaging of Viral Particles
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To examine the morphology of purified viral particles, the purified fresh virion preparations were loaded onto carbon-coated copper grids (200-mesh) for a 5 min incubation at room temperature and negatively stained with 3% (w/v) phosphotungstic acid (pH 6.8) for 1 min. After drying overnight, all the sample grids were examined using TEM (Hitachi, Tokyo, Japan, H-7000FA). For cryo-EM, the prepared viral specimen (~3.0 μL/each) was applied to one side of a holey carbon grid and then quickly plunged into a bath of liquid ethane cooled by liquid nitrogen using an FEI Vitrobot (Thermo Fisher Scientific, MA, USA). The frozen-hydrated samples were transferred to a precooled GATAN cryo-holder and observed using a JEOL 1200 transmission electron microscope operated at 200 kV and maintained at liquid nitrogen temperature.
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