key: cord-0959657-o8oous9n
authors: Du, Shuo; Liu, Pulan; Zhang, Zhiying; Xiao, Tianhe; Yasimayi, Ayijiang; Huang, Weijin; Wang, Youchun; Cao, Yunlong; Xie, Xiaoliang Sunney; Xiao, Junyu
title: Structures of SARS-CoV-2 B.1.351 neutralizing antibodies provide insights into cocktail design against concerning variants
date: 2021-08-25
journal: Cell Res
DOI: 10.1038/s41422-021-00555-0
sha: ed9b9eb8f4486e7d115dcb1158ea8a74f12759cb
doc_id: 959657
cord_uid: o8oous9n

nan

S6P(B.1.351) plasmid was transfected into the HEK293F cells using polyethylenimine (Polysciences). The S6P(B.1.351) protein was retrieved from the conditioned media using the Ni-NTA resin (GE Life Sciences), and then further purified using a Superose 6 increase gel filtration column (GE Life Sciences) in the final buffer (20 mM HEPES, pH 7.2, and 150 mM NaCl). To obtain the antibody Fab fragments, the plasmids encoding the heavy chain and light chain regions were co-transfected into the HEK293F cells. A C-terminal His6 tag is present on the heavy chain. The Fabs were also purified from the conditioned media using Ni-NTA resin, and then passed through a Superdex 200 increase gel filtration column and eluted using the final buffer.

To prepare the sample for cryo-EM study, four microliter S6P(B.1.351) protein (0.7 mg/mL) was mixed with the same volume of indicated Fabs (1 mg/mL each) , and immediately applied onto the glow-discharged holy-carbon gold grids (Quantifoil, R1.2/1.3) in an FEI Vitrobot IV (4 °C and 100% humidity), before the grids were plunged into the liquid ethane. Afterwards, the grids were screened using a Talos Arctica (operated at 200 kV). Good grids were then transferred to a Titan Krios (operating at 300 kV) equipped with a K3 direct detection camera (Gatan) for data collection.

All the image processing procedures were performed using cryoSPARC 5 . Driftcorrection and electron dose-weighting were applied to the movie stacks using the Patch motion correction (multi) module. The contrast transfer function (CTF) parameters were estimated for each summed image using the Patch CTF estimation (multi) module. Micrographs were manually screened within the Manually Curate Exposures module and qualified micrographs were selected for further processing.

Particle picking was carried out using Blob picker and Template picker, and extracted particles were subjected to reference-free 2D classification to discard noise and junk particles. Good particles were selected and used for Ab-Initio Reconstruction and Heterogeneous Refinement. The particles in qualified groups were then gathered and subjected to Homogeneous Refinement. To improve the local resolution around Fab-RBD interface, local refinement was performed with a soft mask applied to encompass the Fabs and RBD region. The mask was created using UCSF Chimera 6 and Relion 7 .

Local resolution map was calculated using cryoSPARC and displayed using UCSF Chimera. Structure modeling and refinement were performed using Coot 8 and Phenix 9 . 

Quantification of SARS-CoV-2 neutralizing antibody by a