Negative-Stain SARS-CoV-2 S-ACE2 Sample Imaging

For the negative-stain sample, a volume of 5 μl of SARS-CoV-2 S-ACE2 sample was placed on a plasma-cleaned copper grid for 1 minute. Excess sample on the grid was blotted off using filter paper, and a volume of 5 μl of 0.75% uranyl formate (UF) (Sigma-Aldrich) was added to wash the grid. After blotting, another volume of 5 μl of 0.75% UF was placed on the grid again for 1 minute to stain. Grids were visualized under a Tecnai G2 Spirit 120-kV transmission electron microscope (Thermo Fisher Scientific), and micrographs were taken using an Eagle camera with a nominal magnification of 67,000 ×, yielding a pixel size of 1.74 Å. A total of 41,827 particles were autopicked in EMAN2 (48 (link)). After 2D classification, we selected good averages with 13,047 particles for initial model building, which were performed in Relion 3.0 (49 (link)).

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Xu C., Wang Y., Liu C., Zhang C., Han W., Hong X., Wang Y., Hong Q., Wang S., Zhao Q., Wang Y., Yang Y., Chen K., Zheng W., Kong L., Wang F., Zuo Q., Huang Z, & Cong Y. (2021). Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM. Science Advances, 7(1), eabe5575.

Publication 2021

Tecnai G2 Spirit 120-kV transmission electron microscope

A 827 Ace2 Copper Eagle Paper Plasma Sars cov 2 Stain Transmission electron microscope Uranyl formate

Corresponding Organization : Institut Pasteur of Shanghai

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Variable analysis

independent variables

Volume of SARS-CoV-2 S-ACE2 sample placed on grid
Duration of SARS-CoV-2 S-ACE2 sample incubation on grid
Concentration of uranyl formate (UF) solution
Duration of UF staining

dependent variables

Number of particles autopicked in EMAN2
Number of particles selected for initial model building

control variables

Type of grid (copper)
Grid preparation (plasma-cleaned)
Transmission electron microscope (Tecnai G2 Spirit 120-kV)
Magnification (67,000x)
Pixel size (1.74 Å)
Software used for image processing (EMAN2, Relion 3.0)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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