Purified SARS-CoV-2 spike ectodomains were diluted to a concentration of ~ 1.5 mg/mL in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3 and 0.5% glycerol was added. A 2.4-μL drop of protein was deposited on a Quantifoil-1.2/1.3 grid (Electron Microscopy Sciences, PA) that had been glow discharged for 10 s using a PELCO easiGlow Glow Discharge Cleaning System. After a 30 s incubation in >95% humidity, excess protein was blotted away for 2.5 s before being plunge frozen into liquid ethane using a Leica EM GP2 plunge freezer (Leica Microsystems). Frozen grids were imaged using a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan). The cryoSPARC (53 (link)) software was used for data processing. Phenix (54 (link), 55 (link)), Coot (56 (link)), Pymol (57 ), Chimera (58 (link)), ChimeraX (59 (link)) and Isolde (60 (link)) were used for model building and refinement.
Malewana R.D., Stalls V., May A., Lu X., Martinez D.R., Schäfer A., Li D., Barr M., Sutherland L.L., Lee E., Parks R., Beck W.E., Newman A., Bock K.W., Minai M., Nagata B.M., DeMarco C.T., Denny T.N., Oguin TH I.I.I., Rountree W., Wang Y., Mansouri K., Edwards R.J., Sempowski G.D., Eaton A., Muramatsu H., Henderson R., Tam Y., Barbosa C., Tang J., Cain D.W., Santra S., Moore I.N., Andersen H., Lewis M.G., Golding H., Seder R., Khurana S., Montefiori D.C., Pardi N., Weissman D., Baric R.S., Acharya P., Haynes B.F, & Saunders K.O. (2023). Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates. bioRxiv.
Other organizations :
International Vaccine Institute, Duke University, University of North Carolina at Chapel Hill, Institute of Infection and Immunity, National Institute of Allergy and Infectious Diseases, National Institutes of Health, University of Pennsylvania, Acuitas Therapeutics (Canada), Center for Biologics Evaluation and Research, Food and Drug Administration, Hadassah Medical Center, Bioqual
Concentration of purified SARS-CoV-2 spike ectodomains (diluted to ~1.5 mg/mL)
dependent variables
Cryo-EM imaging of the purified SARS-CoV-2 spike ectodomains
control variables
2 mM Tris pH 8.0
200 mM NaCl
0.02% NaN3
0.5% glycerol
Quantifoil-1.2/1.3 grid
10 s glow discharge of the grid
30 s incubation in >95% humidity
2.5 s blotting time
Plunge freezing in liquid ethane
Titan Krios cryo-EM microscope with K3 detector
CryoSPARC software for data processing
Phenix, Coot, Pymol, Chimera, ChimeraX, and Isolde for model building and refinement
positive controls
Not explicitly mentioned
negative controls
Not explicitly mentioned
Annotations
Based on most similar protocols
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