Cryo-electron microscopy was performed as in (35 (link),36 (link)). In brief, images (electron dose of ~25 e−/Å2;−4 to−6 µm defocus) were acquired at 27,500×on a Tecnai F30 electron microscope (FEI Company Ltd.) operated at 300 kV. The detector was a GATAN UltraCam, lens-coupled, 4 K CCD camera (binned by 2) attached to a Tridiem Gatan Image Filter (GIF: operated at zero-loss mode with an energy window of 20 eV; Gatan, Inc., Pleasanton, CA, USA). For cryo-electron tomography, tilt series were acquired at−4 or−6 µm defocus, every 1.5 degree (±60 degrees) using serialEM software (37 (link)). The total electron dose was kept at 80–120 e−/Å2.
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Cryo-Electron Microscopy Protocols for Structural Analysis
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Cryo electron microscopy
Electron
Electron microscope
Electron tomography
Lens
Corresponding Organization :
Other organizations : University of Gothenburg
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Variable analysis
independent variables
- Cryo-electron microscopy method
- Electron dose (approximately 25 e-/Å2)
- Defocus (-4 to -6 μm)
- Magnification (27,500x)
- Electron microscope and detector specifications (Tecnai F30, GATAN UltraCam)
- Electron microscope operation at 300 kV
- GATAN Image Filter operation in zero-loss mode with 20 eV energy window
- Cryo-electron tomography tilt series acquisition (every 1.5 degree, ±60 degrees)
- Total electron dose for cryo-electron tomography (80-120 e-/Å2)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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