Cryo-ET Imaging of Protein Particles

Cryo-ET tilt series were acquired using SerialEM software^{53 (link)} from +60° to −60° with a step of 3°, on an FEI Titan Krios microscope (300 kV) equipped with a Gatan K2 camera. At each tilt angle, we collected movies containing 8 frames, with a sum dose of ~3 e⁻ Å⁻² s⁻¹, and the total dose for every tilt series (+60° to −60°) was 120 e⁻ Å⁻². The pixel size was 1.25 Å. The movies were first motion-corrected by MotionCor2^{47 (link)} and then imported into Etomo^{54 (link)} for alignment and reconstruction. The position of protein particles inside the ice was manually identified and plotted^{13 (link)}.

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Zheng L., Liu N., Gao X., Zhu W., Liu K., Wu C., Yan R., Zhang J., Gao X., Yao Y., Deng B., Xu J., Lu Y., Liu Z., Li M., Wei X., Wang H.W, & Peng H. (2022). Uniform thin ice on ultraflat graphene for high-resolution cryo-EM. Nature Methods, 20(1), 123-130.

Publication 2022

Titan Krios microscope K2 camera

Frames Microscope Protein Reconstruction

Corresponding Organization : Tsinghua University

Other organizations : Beijing National Laboratory for Molecular Sciences, Peking University, Hainan Medical College Hospital, Southern University of Science and Technology, Beijing Graphene Institute

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Variable analysis

independent variables

Tilt angle of the cryo-ET sample from +60° to -60° with a step of 3°

dependent variables

Position of protein particles inside the ice

control variables

Microscope (FEI Titan Krios, 300 kV)
Camera (Gatan K2)
Electron dose rate (~3 e- Å-2 s-1)
Total electron dose (120 e- Å-2)
Pixel size (1.25 Å)
Motion correction (MotionCor2)
Alignment and reconstruction (Etomo)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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