Cryo-EM Data Acquisition and Processing

For sample-specific details, refer to Table S6.
Movies were collected in compressed tiff format on a Titan Krios G2 (Thermo Fisher) operating at 300 kV with a K3 detector (Gatan) in super resolution counting mode using a custom version of EPU 2.5 (Thermo Fisher). A defocus range of 0.8-2.6 μm was applied with a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel and with a total dose of 43-47 e/ Å², see Table S6.
Two-times binned movies were then motion corrected and aligned on the fly using Relion(3.1) scheduler (Zivanov et al., 2018 (link)) with a 5 × 5 patch based alignment. CTF-estimation of full-frame non-weighted micrographs was performed with the GCTF (1.06) (Zhang, 2016 (link)) module in cryoSPARC(v2.14.1-live) (Punjani et al., 2017 (link)).

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Dejnirattisai W., Zhou D., Ginn H.M., Duyvesteyn H.M., Supasa P., Case J.B., Zhao Y., Walter T.S., Mentzer A.J., Liu C., Wang B., Paesen G.C., Slon-Campos J., López-Camacho C., Kafai N.M., Bailey A.L., Chen R.E., Ying B., Thompson C., Bolton J., Fyfe A., Gupta S., Tan T.K., Gilbert-Jaramillo J., James W., Knight M., Carroll M.W., Skelly D., Dold C., Peng Y., Levin R., Dong T., Pollard A.J., Knight J.C., Klenerman P., Temperton N., Hall D.R., Williams M.A., Paterson N.G., Bertram F.K., Siebert C.A., Clare D.K., Howe A., Radecke J., Song Y., Townsend A.R., Huang K.Y., Fry E.E., Mongkolsapaya J., Diamond M.S., Ren J., Stuart D.I, & Screaton G.R. (2021). The antigenic anatomy of SARS-CoV-2 receptor binding domain. Cell, 184(8), 2183-2200.e22.

Publication 2021

Titan Krios G2 K3 detector

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Corresponding Organization :

Other organizations : University of Oxford, Wellcome Centre for Human Genetics, Diamond Light Source, Washington University in St. Louis, Oxford University Hospitals NHS Trust, Medawar Building for Pathogen Research, MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, Public Health England, Oxford BioMedica (United Kingdom), Worthing Hospital, Medway School of Pharmacy, University of Kent, Chang Gung Memorial Hospital, Chang Gung University, Siriraj Hospital, Mahidol University, Instruct-ERIC

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Variable analysis

independent variables

Defocus range (0.8-2.6 μm)
Total dose (43-47 e/ Å^2)

dependent variables

Image quality (motion corrected and aligned movies)
CTF-estimation of full-frame non-weighted micrographs

control variables

Electron microscope (Titan Krios G2)
Electron detector (K3 detector)
Data collection mode (super resolution counting mode)
Data collection software (custom version of EPU 2.5)
Magnification (nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel)

controls

No explicit positive or negative controls mentioned

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