Cryo-EM Structural Analysis of SARS-CoV-2 Fab Complex

A three-fold molar excess of Fab was incubated with SARS-2 CoV 6P Mut7 for 30 minutes at room temperature and deposited on a glow discharged carbon coated Cu grid. The complexes were stained with 2% uranyl formate (w/v) for 90 seconds. An FEI Tecnai Spirit at 120 keV paired with an FEI Eagle 4k x 4k CCD camera was used for data collection, and automated using the Leginon software [44 (link)]. Raw micrographs were stored in the Appion database [45 (link)]. Particles were picked with DogPicker [46 (link)] and data was processed in RELION 3.0 [47 (link)]. Figs were generated using UCSF Chimera [48 (link)].

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Hingankar N., Deshpande S., Das P., Rizvi Z.A., Wibmer C.K., Mashilo P., Ansari M.Y., Burns A., Barman S., Zhao F., Mukherjee S., Torres J.L., Chattopadhyay S., Mehdi F., Sutar J., Rathore D.K., Pargai K., Singh J., Sonar S., Jakhar K., Dandotiya J., Bhattacharyya S., Mani S., Samal S., Singh S., Kshetrapal P., Thiruvengadam R., Batra G., Medigeshi G., Ward A.B., Bhatnagar S., Awasthi A., Sok D, & Bhattacharya J. (2022). A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 Delta variant. PLoS Pathogens, 18(4), e1010465.

Publication 2022

Tecnai Spirit Eagle 4k x 4k CCD camera

Carbon Chimera Eagle Figs Molar Sars cov 2 Seconds Uranyl formate

Corresponding Organization : International AIDS Vaccine Initiative

Other organizations : National Health Laboratory Service

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Variable analysis

independent variables

Incubation of Fab with SARS-2 CoV 6P Mut7

dependent variables

Binding of Fab to SARS-2 CoV 6P Mut7
Structural characteristics of the Fab-SARS-2 CoV 6P Mut7 complex

control variables

Incubation time (30 minutes)
Incubation temperature (room temperature)
Staining method (2% uranyl formate for 90 seconds)
Imaging equipment (FEI Tecnai Spirit at 120 keV, FEI Eagle 4k x 4k CCD camera)
Image acquisition software (Leginon)

controls

No positive or negative controls were explicitly mentioned.

Annotations

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