CcsBA cryo EM samples were prepared on quantifoil holey carbon grids (R2/2 300 mesh copper) and plunge frozen a Vitrobot Mark IV (ThermoFisher Scientific, Brno, CZ). Prior to plunge freezing the girds were plasma cleaned for 1 minute using a Gatan Solarus 950 (Gatan, Pleasanton, CA). The Vitrobot sample chamber was set to 4°C and 100 % humidity. 3 μL of purified CcsBA at a concentration of 2.8 mg/ml was applied to the plasma cleaned quantifoil grids and allowed to incubate for 20 seconds. Grids were then blotted for 2 seconds at a blot force of −1 and plunge frozen into liquid ethane. Vitrified samples were then imaged using a Cs-corrected Thermo Fisher Titan Krios G3 cryo-electron microscope (ThermoFisher Scientific, Eindhoven, NL) operating at an accelerating voltage of 300 kV equipped with a Gatan K2-Summit detector (Gatan, Pleasanton, CA) placed on a BioQuantum 968 GIF-quantum energy filter (Gatan, Pleasanton, CA) operating with a slit width of 20 eV. Data acquisition was automated using the EPU software (ThermoFisher Scientific, Brno, CZ) at a magnification of 105,000× which corresponds to a pixel size of 1.1 Å. Micrograph movies were recorded for 8s with 40 frames. This results in a frame rate of 0.2 sec per frame with a dose rate of 1.65 electrons per Å2 (link) per frame (a total dose of 66 electrons per Å2 (link)). The defocus was varied between −1 to −2.5 μm.
Gatan solarus 950
Manufactured by Ametek
The Gatan Solarus 950 is a high-performance plasma cleaning system designed for preparing samples for electron microscopy. It utilizes a controlled plasma environment to remove organic contaminants from the surface of specimens, improving the quality of electron microscope images.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (6 mentions)
Titan krios g3, by Thermo Fisher Scientific (2 mentions)
Gatan k2 summit detector, by Ametek (2 mentions)
Bioquantum 968 gif quantum energy filter, by Ametek (2 mentions)
Epu software, by Thermo Fisher Scientific (2 mentions)
Titan krios g1, by Thermo Fisher Scientific (1 mentions)
Gatan microscopy suite, by Ametek (1 mentions)
3 protocols using gatan solarus 950
1
Cryo-EM Imaging of CcsBA Protein
2
Cryo-EM Imaging of CcsBA Protein
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CcsBA cryo EM samples were prepared on quantifoil holey carbon grids (R2/2 300 mesh copper) and plunge frozen a Vitrobot Mark IV (ThermoFisher Scientific, Brno, CZ). Prior to plunge freezing the girds were plasma cleaned for 1 minute using a Gatan Solarus 950 (Gatan, Pleasanton, CA). The Vitrobot sample chamber was set to 4°C and 100 % humidity. 3 μL of purified CcsBA at a concentration of 2.8 mg/ml was applied to the plasma cleaned quantifoil grids and allowed to incubate for 20 seconds. Grids were then blotted for 2 seconds at a blot force of −1 and plunge frozen into liquid ethane. Vitrified samples were then imaged using a Cs-corrected Thermo Fisher Titan Krios G3 cryo-electron microscope (ThermoFisher Scientific, Eindhoven, NL) operating at an accelerating voltage of 300 kV equipped with a Gatan K2-Summit detector (Gatan, Pleasanton, CA) placed on a BioQuantum 968 GIF-quantum energy filter (Gatan, Pleasanton, CA) operating with a slit width of 20 eV. Data acquisition was automated using the EPU software (ThermoFisher Scientific, Brno, CZ) at a magnification of 105,000× which corresponds to a pixel size of 1.1 Å. Micrograph movies were recorded for 8s with 40 frames. This results in a frame rate of 0.2 sec per frame with a dose rate of 1.65 electrons per Å2 (link) per frame (a total dose of 66 electrons per Å2 (link)). The defocus was varied between −1 to −2.5 μm.
3
Cryo-EM Imaging of Augmin TIII Tetramer
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4 µl of purified augmin TIII tetramer were applied on Cu R2/1 holey carbon grids (200 mesh; Quantifoil Micro Tools, GmbH) with or without a layer of continuous carbon (2 nm). Grids were glow discharged beforehand using a Gatan Solarus 950 (Gatan, Inc.) plasma cleaner for 30 s. The grids were blotted for 0.5 s at room temperature and 85% relative humidity and plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Screening of the grids showed better distribution of particles on grids with continuous carbon layer, which accordingly were selected for high-resolution data acquisition. Data were acquired in one session on a Titan Krios G1 (Thermo Fisher Scientific) operated at 300 kV, equipped with a K3 camera operated by Gatan Microscopy Suite (version 3.32, Gatan, Inc.) and a Quanta energy filter. Data were acquired in dose fractionation mode (30 frames) at a pixel size of 1.07 Å with a cumulative dose of 68.9 e-/Å2 and a dose rate of 27.9 e-/px/s. Data were acquired in EPU (version 2.6, Thermo Fisher Scientific) using aberration free image shift (AFIS) with 4 images per hole and a nominal defocus range of −1 to −3 µm.
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