Initial screening of grids was performed in Sheffield on Tecnai Arctica (FEI) 200kV cryo-electron microscope. Two separate data collections were done from the same grid using a Titan Krios microscope (FEI) operated at 300 keV and equipped with a Gatan K2-Summit detector and energy filter (a slit width of 20 eV was used) at Astbury Biostructure Laboratory, University of Leeds. Movies were collected automatically with EPU (Thermo Fisher Scientific) software on Gatan K2-Summit detector operated in counting mode with calibrated pixel size of 1.07 Å. For the 1st dataset 682 movies were collected with target defocus range of 1.3 to 3.1 μm. Each movie comprised 50 frames with a total fluence of 53 e-/Å2 over 8 s, corresponding to a flux of 7.58 e-/pixel/s. For the 2nd data collection 2085 movies were collected over a target defocus range of 1.0 to 2.4 μm with a total fluence of 54 e-/Å2 over 8 s, corresponding to a flux of 7.65 e-/pixel/s. To address any preferential orientation problem 995 movies were collected at 10° stage tilt and 71 at 20° stage tilt.
Gatan k2 summit detector
Manufactured by Thermo Fisher Scientific
The Gatan K2-Summit detector is a direct electron detector designed for cryo-electron microscopy applications. It captures high-resolution images with high contrast and low noise, enabling detailed structural analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Titan krios microscope, by Thermo Fisher Scientific (2 mentions)
R1 2 1, by Quantifoil (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Tecnai arctica microscope, by Thermo Fisher Scientific (1 mentions)
Falcon 2 4k 4k camera, by Thermo Fisher Scientific (1 mentions)
Cs corrector, by Thermo Fisher Scientific (1 mentions)
2 protocols using gatan k2 summit detector
1
Cryo-EM Data Collection Workflow
2
Cryo-EM Sample Preparation and Imaging
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The cryo-EM grids were prepared using a Vitrobot Mark IV (FEI) operated at 8 °C and 100% humidity. For samples of hNKCC1 and mKCC2, 4 μl aliquots of fresh samples at concentrations of ~10 mg/ml were applied onto glow-discharged holey carbon grids, 300 mesh gold (Quantifoil R1.2/1.3). After 5 s, the grids were blotted for 2 s and plunged into liquid ethane for quick freezing.
The cryo-EM grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II 4k × 4k camera (FEI). Qualified grids were transferred to a Titan Krios microscope (FEI) operated at 300 kV for data acquisition and equipped with Cs-corrector (ThermoFisher Scientific Inc.), Gatan K2 Summit detector, and GIF Quantum energy filter. Images were automatically recorded using AutoEMation with a slit width of 20 eV for the energy filter and in super-resolution mode at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 1.091 Å at object scale, and with defocus ranging from 1.4 to 1.9 μm. Each stack was exposed for a total of 5.6 s with an exposure of 0.175 s for each of 32 frames—the total dose for each stack was about 50 e−/Å2.
The cryo-EM grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II 4k × 4k camera (FEI). Qualified grids were transferred to a Titan Krios microscope (FEI) operated at 300 kV for data acquisition and equipped with Cs-corrector (ThermoFisher Scientific Inc.), Gatan K2 Summit detector, and GIF Quantum energy filter. Images were automatically recorded using AutoEMation with a slit width of 20 eV for the energy filter and in super-resolution mode at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 1.091 Å at object scale, and with defocus ranging from 1.4 to 1.9 μm. Each stack was exposed for a total of 5.6 s with an exposure of 0.175 s for each of 32 frames—the total dose for each stack was about 50 e−/Å2.
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