Holey carbon grids (Quantifoil Au 300 mesh, R 0.6/1) were glow-discharged by Solarus advanced plasma system (Gatan) for 120 s using 25% O2 and 75% Ar. Aliquots of 3 μl concentrated protein sample were applied on glow-discharged grids and the grids were blotted for 2 s before plunged into liquid ethane using Vitrobot Mark IV (ThermoFisher Scientific).
Au 300 mesh
Manufactured by Quantifoil
The Quantifoil Au 300 mesh is a laboratory equipment product. It is a thin gold-coated polymer film with a regular array of precisely spaced holes, designed for use in electron microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (6 mentions)
Solarus advanced plasma system, by Ametek (2 mentions)
K3 summit detector, by Ametek (1 mentions)
300 kv titan krios, by Thermo Fisher Scientific (1 mentions)
Talos arctica, by Thermo Fisher Scientific (1 mentions)
Solarus 2, by Ametek (1 mentions)
Epu software, by Thermo Fisher Scientific (1 mentions)
Krios tem, by Ametek (1 mentions)
6 protocols using au 300 mesh
1
Cryo-EM Sample Preparation Protocol
2
Cryo-EM Sample Preparation Protocol
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Holey carbon grids (Quantifoil Au 300 mesh, R 0.6/1) were glow-discharged by Solarus advanced plasma system (Gatan) for 120 s using 50% O 2 and 50% Ar. Aliquots of 3μl concentrated protein sample were applied on glow-discharged grids and the grids were blotted for 2s before plunged into liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific).
3
Cryo-EM Sample Preparation for SIDT2 Proteins
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Holey carbon grids (Quantifoil Au 300 mesh, R1.2/1.3) were glow-discharged in the Plasma Cleaner PDC-32G-2 (Harrick Plasma Company) with a vacuum for 2 min and mid force for 30 s. Aliquots (4 µl) of SIDT2 proteins were placed on the glow-discharged grids, which were then blotted for 3 s and flash frozen in liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific) at 8 °C and 100% humidity. The grids were loaded onto a 300 kV Titan Krios (Thermo Fisher Scientific Inc.) equipped with K3 Summit detector (Gatan) and GIF Quantum energy filter. Images were automatically collected using AutoEMation49 (link) in super-resolution mode at a nominal magnification of 81,000 × (64,000 × for apoSIDT2-pH 7.4 dataset), with a slit width of 20 eV on the energy filter. A defocus series ranging from −1.3 μm to −1.8 μm was used. Each stack was exposed for 2.56 s with an exposure time of 0.08 s per frame, resulting in a total of 32 frames per stack and the total dose was approximately 50 e-/Å2 for each stack. The stacks were motion corrected with MotionCor250 (link) and binned 2 fold, resulting in a pixel size of 1.0825 Å/pixel (1.0979 Å for apoSIDT2-pH 7.4 dataset). Meanwhile, dose weighting was performed51 (link). The defocus values were estimated with Gctf52 (link).
4
Preparation of Graphene-Coated Cryo-EM Grids
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Here, 3.5 μl (7 optical density (OD260nm)/ml) of each antibiotic–ribosome complex 1–5 were applied to freshly plasma-cleaned graphene coated TEM grids (Quantifoil, Au, 300 mesh, R1.2/1.3). Graphene coating was carried out according to the in-house optimized protocol for transfer of monolayer chemical vapor deposition graphene to the grid surface. The graphene grids were then hydrophilized in H:O plasma (40:1) for 30 s using Gatan Solarus II immediately before use. Sample vitrification into liquid ethane was performed using a Thermo Scientific Vitrobot Mark IV (4 °C, 100% rel. humidity, 30 s waiting time, 3 s blotting time). The grids were subsequently mounted into the Autogrid cartridges and loaded to Talos Arctica (Thermo Scientific) TEM for screening. Grids were stored in liquid nitrogen until high-resolution data collection.
5
Cryo-EM Sample Preparation and Imaging
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3-μl of the sample was applied to a glow-discharged grid (Quantifoil Au 300 mesh, R1.2/1.3), blotted for 2 seconds and rapidly plunged into liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific) operating at 8 °C and 100% humidity. Cryo-EM samples were imaged on a 300-kV Titan Krios electron microscope using a nominal magnification of 81,000×. Movies were recorded using a Gatan K3 detector equipped with a GIF Quantum energy filter (slit width 20 eV) at the counted mode, with a pixel size of 0.5435 Å. Data collection were performed using EPU software (Thermo Fisher Scientific). Each stack of 32 frames was exposed for 2.56 seconds, with a dose rate of ~23 counts/second/pixel for each frame. All 32 frames in each stack were aligned and summed using MotionCor254 (link) and binned to a pixel size of 1.087 Å. The defocus value for each image varied from −1.5 to −2.0 μm and was determined by Gctf55 (link).
6
Cryo-EM Imaging of PKD1-PKD2 Complex
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Holey carbon grids (Quantifoil Au 300 mesh, R1.2/1.3) were glow-discharged in the Plasma Cleaner PDC-32G-2 (HARRICK PLASMA Company) with a vacuum for 2 min and mid force for 22 s. Aliquots (3 ml) of purified PKD1-PKD2 complex at concentration of ~10 mg/ml were placed on the glow-discharged grids, which were then blotted for 2.5 to 3.5 s and flash frozen in liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific) at 8°C and 100% humidity without wait time or blot force.
The grids were transferred to a Titan Krios TEM operated at 300 kV and equipped with Gatan GIF Quantum energy filter and Gatan K2 direct electron detector and Cs corrector. A total of 3761 zero-loss movie stacks were automatically collected using AutoEMation II (developed by J. Lei) in the super-resolution mode (62) (link) with 20-eV slit in energy filter at a nominal magnification of 105,000× with defocus range from -1.0 to -2.0 mm. Each micrograph stack, which contained 32 frames, was exposed for 5.6 s with a total electron dose of ~50 e -/Å 2 . The stacks were motion corrected using MotionCor2 (63) with a binning factor of 2, resulting in a pixel size of 1.091 Å. Dose weighting was performed concurrently (64) (link). The defocus values were estimated with Gctf (65) (link).
The grids were transferred to a Titan Krios TEM operated at 300 kV and equipped with Gatan GIF Quantum energy filter and Gatan K2 direct electron detector and Cs corrector. A total of 3761 zero-loss movie stacks were automatically collected using AutoEMation II (developed by J. Lei) in the super-resolution mode (62) (link) with 20-eV slit in energy filter at a nominal magnification of 105,000× with defocus range from -1.0 to -2.0 mm. Each micrograph stack, which contained 32 frames, was exposed for 5.6 s with a total electron dose of ~50 e -/Å 2 . The stacks were motion corrected using MotionCor2 (63) with a binning factor of 2, resulting in a pixel size of 1.091 Å. Dose weighting was performed concurrently (64) (link). The defocus values were estimated with Gctf (65) (link).
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