PhnG2H2I2J2K was diluted to 0.1 mg/mL with the dilution buffer (50 mM HEPES, pH 8.5, 150 mM NaCl, 2 mM TCEP). 3 μL of the sample was applied to a C-Flat 1.2/1.3 holey carbon grid at 16°C with 100% relative humidity and vitrified using a Vitrobot Mark III (FEI company, Netherlands). The thin-ice areas that were expected to show clearly visible and mono-dispersed particles were imaged under an FEI Tecnai F20 cryo electron microscope with a field emission gun (FEI company, Netherlands) operated at 200 kV. Data were recorded on a Gatan K2 Summit electron-counting direct detection camera (Gatan, Pleasanton CA) in the electron counting mode (Li et al., 2013 (link)). A nominal magnification of 25,000X was used, yielding a pixel size of 1.5 Å. The beam intensity was adjusted to a dose rate of ~10 electrons per pixel per second on the camera. A 25-frame or 50-frame movie stack was recorded for each picture, with 0.2 second per frame for a total exposure time of 5 seconds or 10 seconds, respectively.
K2 summit electron counting direct detection camera
Manufactured by Ametek
The K2 Summit electron-counting direct detection camera is a high-performance imaging device designed for use in electron microscopy applications. It features a direct detection sensor that can count individual electrons, enabling high-resolution imaging with improved signal-to-noise ratio.
Automatically generated - may contain errors
2 protocols using k2 summit electron counting direct detection camera
1
Cryo-EM Imaging of PhnG2H2I2J2K
2
Cryo-EM Imaging Using Leica EM GP and Titan Krios
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cryo-EM grids were prepared using a Leica EM GP automatic plunge freezer (Leica Microsystems) with 80% humidity at 20°C. 3 µL aliquots were applied onto 60 s glow-discharged, 300 mesh, copper Quantifoil grids (R2/1). After blotting, grids were plunge frozen in liquid ethane cooled by liquid nitrogen.
Micrographs were acquired on a Titan Krios (ThermoFisher Scientific) transmission electron microscope, operated at 300 kV and equipped with a Gatan Quantum-LS imaging energy filter (GIF, 20 eV zero loss energy window; Gatan Inc). Images were recorded on a K2 Summit electron counting direct detection camera (Gatan Inc) in dose fractionation mode (50 frames) using the Serial EM software (Mastronarde, 2005 (link)) at pixel sizes of 0.831 Å or 0.629 Å, and a total dose of ~69 electrons per square Angstrom (e-/Å2) for each micrograph. Micrographs were processed and analyzed during data collection with FOCUS (Biyani et al., 2017 (link)), applying drift-correction and dose-weighting using MotionCor2 (Zheng et al., 2017 (link)). Specific data collection parameters for the various datasets are detailed inTable 2 .
Micrographs were acquired on a Titan Krios (ThermoFisher Scientific) transmission electron microscope, operated at 300 kV and equipped with a Gatan Quantum-LS imaging energy filter (GIF, 20 eV zero loss energy window; Gatan Inc). Images were recorded on a K2 Summit electron counting direct detection camera (Gatan Inc) in dose fractionation mode (50 frames) using the Serial EM software (Mastronarde, 2005 (link)) at pixel sizes of 0.831 Å or 0.629 Å, and a total dose of ~69 electrons per square Angstrom (e-/Å2) for each micrograph. Micrographs were processed and analyzed during data collection with FOCUS (Biyani et al., 2017 (link)), applying drift-correction and dose-weighting using MotionCor2 (Zheng et al., 2017 (link)). Specific data collection parameters for the various datasets are detailed in
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