key: cord-0843649-mj3djxy9
authors: Wang, Nan; Sun, Yao; Feng, Rui; Wang, Yuxi; Guo, Yan; Zhang, Li; Deng, Yong-Qiang; Wang, Lei; Cui, Zhen; Cao, Lei; Zhang, Yan-Jun; Li, Weimin; Zhu, Feng-Cai; Qin, Cheng-Feng; Wang, Xiangxi
title: Structure-based development of human antibody cocktails against SARS-CoV-2
date: 2020-12-01
journal: Cell Res
DOI: 10.1038/s41422-020-00446-w
sha: f4319d7d36e11d6b29590ea57d9cc339d0cc9048
doc_id: 843649
cord_uid: mj3djxy9

nan

(IBA). For further purification, the eluted sample was concentrated and subjected to additional 11 purification by size-exclusion chromatography in 20 mM Tris, 200 mM NaCl, pH 8.0. 12

All the purified Fab fragments and purified SARS-CoV-2 trimer were diluted to 1 mg/ml. FC05 14

Fab fragments were firstly mixed with Fab fragments of H014, HB27 or P17, respectively. Then 15 each mixture was incubated with purified SARS-CoV-2 trimer at a mol ratio of 3:1 at ice for 3 16 min. The C-flat 1.2/1.3 Au grid was glow-discharged and transferred with a 3 μl incubated 17 aliquot. After 3 s blotting in 100% relative humidity, the grid was plunged into liqu id ethane in 18

Vitrobot (Thermo Fisher Scientific). All three Cryo-EM datasets were collected at 300 kV using 19

Titan Krios microscope (Thermo Fisher Scientific) equipped with a K2 detector (Gatan, 20

Pleasanton, CA). Movies were recorded at 32 frames, with total dose of 60 e -Å -2 and a 1.5-2.7 21 μm defocus using serialEM yielding the final pixel size of 1.04 Å. 22

Totally 1,398 stacks for FC05-P17-S complex, 1,021 stacks for FC05-H014-S complex, and 23 1,532 stacks for FC05-HB27-S complex were recorded. RelionCorr 12 was used to correct beam 24 induced motion and average frames. The GPU accelerated Gctf 13 was used to estimate CTF Table S1 . 39

SARS-CoV-2 S trimer was immobilized onto a CM5 sensor chip surface using the NHS/EDC 41 method to a level of ~1,800 response units (RUs) using Bia-core T100 (GE Healthcare) and a 42 PBS running buffer (suspended with 0.05% Tween-20). Purified FC05, H014, HB27 and P17 43 were prepared for the competitive binding assays. The first sample FC05 flew over the chip at a 44 rate of 20 ul/min for 120s, then the other three different antibodies individually were injected at 45 the same rate for another 120s. The FC05 antibody was used as a negative control. All antibodies 46 were evaluated at saturation concentration of 500 nM, except for FC05 (1000 nM). All antibodies 47 were regenerated with 35 mM NaOH. The response of the three antibodies to the FC05 was 48 recorded at room temperature and the data was analyzed using Bia-core T100 Evaluation 49 Software (GE Healthcare). 

Cryo-EM 65 maps of FC05-H014-S (a), FC05-HB27-S (b) and FC05-P17-S complexes (c) are shown. The right 66 panels present the density maps (mesh) and related atomic models shown as sticks

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Processing of Structurally Heterogeneous Cryo-EM Data in RELION

Real-time CTF determination and correction

Quantifying the local resolution of cryo-EM density 82 maps

UCSF Chimera, MODELLER, and IMP: an integrated modeling system

Coot: model-building tools for molecular graphics

Towards automated crystallographic structure refinement with phenix. 88 refine

MolProbity: all-atom structure validation for macromolecular crystallography