Cryo-EM Structural Analysis of S Protein Trimer

S protein trimer with D614G mutation was prepared as described previously^{26 (link)} and mixed with 1:1 molar ratio of NIBIC-71 and 7G7/κ solutions at a final concentration of 0.5 mg/mL of the S protein trimer. Quantifoil grids (R1.2/1.3 Cu 200 mesh) were glow-discharged using a JEC-3000FC sputter coater (JEOL) at 20 mA for 20 s. After 3 mL of the complex solutions were applied, the grids were blotted with a force of –10 and a time of 2 s in a Vitrobot Mark IV chamber (Thermo) equilibrated at 4 °C and 100% humidity, and then immediately plunged into liquid ethane. The grids were stored in liquid nitrogen. All cryo-EM image datasets were acquired using SeriaIEM^{27 (link)}, yoneoLocr^{28 (link)}, and a JEM-3300 (CRYO ARM™ 300 II, JEOL) operated at 300 kV with a K3 direct electron detector (Gatan, Inc.) in CDS mode. The W-type in-column energy filter was operated with a slit width of 20 eV for zero-loss imaging. The nominal magnification was 60,000×, corresponding to 0.86 Å per pixel. Defocus varied between − 0.5 and − 2.0 mm. Each movie was fractionated into 60 frames with a total dose of 60 e⁻/Å².

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Otsubo R., Minamitani T., Kobiyama K., Fujita J., Ito T., Ueno S., Anzai I., Tanino H., Aoyama H., Matsuura Y., Namba K., Imadome K.I., Ishii K.J., Tsumoto K., Kamitani W, & Yasui T. (2022). Human antibody recognition and neutralization mode on the NTD and RBD domains of SARS-CoV-2 spike protein. Scientific Reports, 12, 20120.

Publication 2022

JEC-3000FC sputter coater Vitrobot Mark IV chamber K3 direct electron detector

Electron Ethane Frames Humidity Molar Mutation Nitrogen S protein

Corresponding Organization :

Other organizations : National Institute of Biomedical Innovation, Health and Nutrition, The University of Tokyo, Osaka University, Gunma University, National Center For Child Health and Development

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Variable analysis

independent variables

The molar ratio of NIBIC-71 and 7G7/κ solutions mixed with the S protein trimer

dependent variables

None explicitly mentioned

control variables

The final concentration of the S protein trimer (0.5 mg/mL)
The Quantifoil grids (R1.2/1.3 Cu 200 mesh) used
The glow-discharge parameters (20 mA for 20 s)
The Vitrobot Mark IV chamber conditions (4 °C and 100% humidity)
The blotting force (-10) and time (2 s)
The cryo-EM imaging parameters (JEM-3300 operated at 300 kV, K3 direct electron detector in CDS mode, W-type in-column energy filter with 20 eV slit width, 60,000× nominal magnification, -0.5 to -2.0 mm defocus, 60 movie frames with 60 e-/Å2 total dose)

controls

None explicitly mentioned

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