Purified human and chicken STING of either the apo or cGAMP-bound form at 4.5 mg/ml was applied to a glow-discharged Quantifoil R1.2/1.3 300-mesh gold holey carbon grid (Quantifoil, Micro Tools), blotted under 100% humidity at 4 °C and plunged into liquid ethane using a Mark IV Vitrobot (FEI). Micrographs were acquired on a Titan Krios microscope (FEI) with a K2 Summit direct electron detector (Gatan) in the super-resolution counting mode, operated at 300 kV using the EPU software (FEI). The slit width of the GIF-Quantum energy filter was set to 20 eV. A Volta phase plate was used to enhance low-resolution features20 (link). Micrographs were dose-fractioned into 20 frames with a total exposure time of 10 s at the dose rate of 2 e− per pixel per second.
Protocol detail
Cryo-EM Structural Analysis of STING Protein
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Other organizations : The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- Purified human and chicken STING of either the apo or cGAMP-bound form
- Micrographs acquired on a Titan Krios microscope with a K2 Summit direct electron detector in the super-resolution counting mode
- Glow-discharged Quantifoil R1.2/1.3 300-mesh gold holey carbon grid
- Blotting under 100% humidity at 4 °C
- Plunging into liquid ethane using a Mark IV Vitrobot
- Operated at 300 kV using the EPU software
- Slit width of the GIF-Quantum energy filter set to 20 eV
- Volta phase plate used to enhance low-resolution features
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