Cryo-EM Structural Analysis of Protein Complexes

Purified complexes were diluted to 0.03 mg/ml using 1X TBS pH 7.4, deposited on glow-discharged carbon coated copper mesh grids, and stained for 90 sec with 2% uranyl formate (w/v). Samples were imaged either on a FEI Tecnai Spirit T12 transmission electron microscope (120 keV, 52,000x mag, 2.06 Å per pixel, -1.5 μm defocus) equipped with an FEI Eagle 4k x 4k CCD camera or a FEI TF20 transmission electron microscope (200 keV, 62,000x mag, 1.78 Å per pixel, -1.5 μm defocus) equipped with a TVIPS TemCam F416 CMOS 4k x 4k camera. Collection of raw micrographs was automated with the Leginon (Suloway et al., 2005 (link)) software and stored in the Appion (Lander et al., 2009 (link)) database. For each mouse group complex, enough micrographs were collected to have ≥ 100k particles for data processing. Particles were picked using DogPicker (Voss et al., 2009 (link)), and further data processing was performed in RELION 3.0 (Scheres, 2012 (link)). For EMPEM with SARS-CoV-2 spike, an initial model was generated from a published SARS-CoV-2 S protein structure (PDB: 6VYB (Walls et al., 2020 (link))) and used during data processing. Map interpretation and segmentation was performed in UCSF Chimera (Pettersen et al., 2004 (link)).

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Tas J.M., Koo J.H., Lin Y.C., Xie Z., Steichen J.M., Jackson A.M., Hauser B.M., Wang X., Cottrell C.A., Torres J.L., Warner J.E., Kirsch K.H., Weldon S.R., Groschel B., Nogal B., Ozorowski G., Bangaru S., Phelps N., Adachi Y., Eskandarzadeh S., Kubitz M., Burton D.R., Lingwood D., Schmidt A.G., Nair U., Ward A.B., Schief W.R, & Batista F.D. (2022). Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses. Immunity, 55(10), 1856-1871.e6.

Publication 2022

Tecnai Spirit T12 Eagle 4k x 4k CCD camera

Carbon Chimera Cmos Copper Eagle Mouse Sars cov 2 Sars cov 2 s protein Transmission electron microscope Uranyl formate

Corresponding Organization : Harvard University

Other organizations : Scripps Research Institute, International AIDS Vaccine Initiative

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Variable analysis

independent variables

Purified complexes were diluted to 0.03 mg/ml using 1X TBS pH 7.4
Samples were imaged either on a FEI Tecnai Spirit T12 transmission electron microscope (120 keV, 52,000x mag, 2.06 Å per pixel, -1.5 μm defocus) or a FEI TF20 transmission electron microscope (200 keV, 62,000x mag, 1.78 Å per pixel, -1.5 μm defocus)

dependent variables

Collection of raw micrographs

control variables

Purified complexes were deposited on glow-discharged carbon coated copper mesh grids
Purified complexes were stained for 90 sec with 2% uranyl formate (w/v)
Initial model generated from a published SARS-CoV-2 S protein structure (PDB: 6VYB) was used during data processing

positive controls

Published SARS-CoV-2 S protein structure (PDB: 6VYB)

negative controls

Not explicitly mentioned

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