Source Data: Structure and inhibition of SARS-CoV-2 spike refolding in membranes

Grunst, Michael 1 ; Qin, Zhuan1; Dodero-Rojas, Esteban2; Ding, Shilei3; Prévost, Jérémie3; Chen, Yaozong4; Hu, Yanping5; Pazgier, Marzena4; Wu, Shenping1; Xie, Xuping5; Finzi, Andrés3; Onuchic, José2; Whitford, Paul6 ; Mothes, Walther1 ; Li, Wenwei1

Research facility: Yale University

Published Sep 05, 2024 on Dryad. https://doi.org/10.5061/dryad.ncjsxkt3h

Data files

Sep 05, 2024 version files 308.84 GB

Grunstetal_Biological_Cryo_PrehairpinModels.zip

3.63 MB
README.md

4.45 KB
SpikeSimsData.tar.gz

308.84 GB

Abstract

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein binds the receptor Angiotensin Converting Enzyme 2 (ACE2) and drives virus-host membrane fusion through refolding of its S2 domain. Whereas the S1 domain contains high sequence variability, the S2 domain is conserved and is a promising pan-betacoronavirus vaccine target. We applied cryo-electron tomography to capture intermediates of S2 refolding and understand inhibition by anti-S2 stem-helix antibodies. Subtomogram averaging revealed ACE2 dimers cross-linking spikes before transitioning into S2 intermediates, which were captured at various stages of refolding. Pan-betacoronavirus neutralizing antibodies targeting the S2 stem-helix bound to and inhibited refolding of spike prehairpin intermediates. Combined with molecular dynamics simulations, these structures elucidate the process of SARS-CoV-2 entry and reveal how pan-betacoronavirus S2-targeting antibodies neutralize infectivity by arresting prehairpin intermediates.

https://doi.org/10.5061/dryad.ncjsxkt3h

This dataset contains excel sheets from biological assays, a summary excel document of all cryoET measurements (Spike angles and membrane distances), scripts used for data processing and visualization, and MD Simulation data. PDB files for simulated prehairpin intermediate atomic models are also included.

Description of the data and file structure

Directory "Grunstetal_Biological_Cryo_PrehairpinModels":

-The folders "Fig1_Virus-CellFusionAssays", "Fig4_pre-post_attachment_assays", and "Fig4_FigS9_Neutralization_CellStaining" include excel sheets from biological assays in this manuscript (pre-post attachment assays, fusion assays, neutralization assays, cell surface staining).

-In the folder "Prehairpin_PDBFiles", PDB files for simulated prehairpin intermediate models shown in Figure 3 and Fig S8 are included (Low Tilt, Medium Tilt, High Tilt). These models and the prefusion S2 (before extension) are also available through (https://smog-server.org/ as previously described in Dodero-Rojas, elife, 2021, https://doi.org/10.7554/eLife.70362). Also included are our MD-Sim_Postfusion Hybrid models shown in Fig. S8. The postfusion component is derived from PDB: 8FDW (https://doi.org/10.1038/s41586-023-06273-4). Construction of these hybrid models has been described in the manuscript text.

-The folder “MembraneDistance_Angles_Timepoints” include an excel document containing all of the membrane distances (in nanometers), angles, and timepoints for the MD ensembles of structures which were used in plotting and statistical tests (i.e. scatter plots, Kruskal-Wallis tests, Mann-Whitney U tests, and mode detection).

Sheet "Membrane Distances" contains all of the membrane distance information for all samples. Related to Fig. 2B and 2C, Fig. 4K and 4L, and fig. S4, along with the R script "MultimodeRscript_share.R" for detecting modes and generating histograms from multimodal distributions for membrane distance analysis (all necessary imported packages are listed in the script).
Sheet "Angle_vs_Distance_Prehairpin" contains all of the prehairpin spike angle information next to the membrane distance values. Note that there are multiple spike angles associated with a single membrane distance (multiple spikes are present at each interface). Related to Fig. 3G and the python script "Angle_Distance_DensityPlot_share.py" for generating the scatter plots, where the points are colored by point density for visualization (all necessary imported packages are listed in the script).
Sheet "Angle_vs_Distance_4C_NoAb" contains Prefusion spike angle information next to the membrane distance values (similar to above). For Prefusion Spikes with no antibody. Related to Fig. 2D and python script "Angle_Distance_DensityPlot_share.py"
Sheet "Angle_vs_Distance_4C_CV325Fab" contains Prefusion spike angle information next to the membrane distance values (similar to above). For Prefusion Spikes in the presence of CV3-25 Fab. Related to Fig. 2D and python script "Angle_Distance_DensityPlot_share.py"
Sheet "Compare_Timepoints_Ensembles" contains the timepoints from which the ensembles corresponding to the density maps in Fig. 3 were found. Related to Fig. 3H.

Directory “SpikeSimsData”

This directory contains all simulated trajectories described in this manuscript. The README file in the main directory describes the organization of the subdirectories. README files in individual directories provide file naming conventions. The file md5sum.txt provides md5sum values for all files in the directory, such that file fidelity may be verified by each user.

**Simulation Analysis Tools:

Simulation input decks and analysis scripts are provided through the git repo https://github.com/Whitford/SpikeSimulations. The repo is associated with the following DOI: **10.5281/zenodo.11081576. Input decks for the Fab-free simulations are released in v1. Upon publication, v2 will be released, which will contain input decks for the +Fab simulations and all analysis (Python) scripts.