Modification:Article Title: Structure of a SARS-CoV-2 spike S2 subunit in a pre-fusion, open conformation.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Dulbecco’s Modified Eagle Medium High Glucose with Glutamax supplement Thermo Fisher Scientific Cat# 10566016 Dulbecco’s phosphate-buffered saline Corning Cat#21-031-CV N-hydroxysulfosuccinimide (Sulfo-NHS) ThermoFisher Scientific Cat# 24510 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Pierce Cat# PG82079 MES Carterra Cat# 3625 1M Ethanolamine, pH 8.5 Carterra Cat# 3638 10% Tween 20 Carterra Cat# 3631 0.2M Sodium Hydroxide Carterra Cat# 3638 Platinum SuperFi II Green PCR Master Mix Invitrogen Cat# 12369050 Platinum II Hot-Start Green PCR Master mix (2X) Thermo fisher Cat# 14001-013 NEBuilder HiFi DNA Assembly master mix NEB Cat# E2621L NEB 5 alpha F’Iq competent E coli NEB Cat# C2992I E-gel96 2% with SYBR safe Invitrogen Cat# G720802 Qiaprep 96 Turbo Miniprep kit Qiagen Cat# 27191 SARS-CoV-2-S2 subunit Spike protein ectodomains with VFLIP mutations and C-terminal Foldon, HRV3C protease cleavage site and twin-strep-tag This study N/A SARS-CoV-2 S-2P This Study N/A OptiPRO SFM Gibco Cat# 12309019 Critical commercial assays TMB substrate kit Peirce Cat# 34021 Plasmid Plus Midi ki Qiagen Cat# 12945 ExpiFectamine CHO Transfection kit ThermoFisher Cat# A29129 Deposited data 6C10 and VFLIP S2-DS5 complex (Cryo-EM) This study EMD-42985 Experimental models: Cell lines 293T cells ATCC Cat# CRL-3216 Vero cells ATCC Cat# CRL-1586 ExpiCHO-S cells Thermo Fisher Scientific Cat# A29127 Experimental models: Organisms/strains K18-hACE2 Jackson Labs Cat# 034860 Recombinant DNA SARS-CoV-2 S2 Subunit with stabilizing disulfide mutations This study N/A Plasmid: 6C10 light chain This study N/A Plasmid: 6C10 heavy chain This study N/A Plasmid: 1A11 light chain This study N/A Plasmid: 1A11 heavy chain This study N/A Plasmid: 6E1 light chain This study N/A Plasmid: 6E1 heavy chain This study N/A Plasmid: 4B3 light chain This study N/A Plasmid: 4B3 heavy chain This study N/A Plasmid: 25.10C light chain Robinson et al. 56 N/A Plasmid: 25.10C heavy chain Robinson et al. 56 N/A (Continued on next page) 14 Cell Reports 44, 116052, August 26, 2025
Saline:Article Title: Structure of a SARS-CoV-2 spike S2 subunit in a pre-fusion, open conformation.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Dulbecco’s Modified Eagle Medium High Glucose with Glutamax supplement Thermo Fisher Scientific Cat# 10566016 Dulbecco’s phosphate-buffered saline Corning Cat#21-031-CV N-hydroxysulfosuccinimide (Sulfo-NHS) ThermoFisher Scientific Cat# 24510 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Pierce Cat# PG82079 MES Carterra Cat# 3625 1M Ethanolamine, pH 8.5 Carterra Cat# 3638 10% Tween 20 Carterra Cat# 3631 0.2M Sodium Hydroxide Carterra Cat# 3638 Platinum SuperFi II Green PCR Master Mix Invitrogen Cat# 12369050 Platinum II Hot-Start Green PCR Master mix (2X) Thermo fisher Cat# 14001-013 NEBuilder HiFi DNA Assembly master mix NEB Cat# E2621L NEB 5 alpha F’Iq competent E coli NEB Cat# C2992I E-gel96 2% with SYBR safe Invitrogen Cat# G720802 Qiaprep 96 Turbo Miniprep kit Qiagen Cat# 27191 SARS-CoV-2-S2 subunit Spike protein ectodomains with VFLIP mutations and C-terminal Foldon, HRV3C protease cleavage site and twin-strep-tag This study N/A SARS-CoV-2 S-2P This Study N/A OptiPRO SFM Gibco Cat# 12309019 Critical commercial assays TMB substrate kit Peirce Cat# 34021 Plasmid Plus Midi ki Qiagen Cat# 12945 ExpiFectamine CHO Transfection kit ThermoFisher Cat# A29129 Deposited data 6C10 and VFLIP S2-DS5 complex (Cryo-EM) This study EMD-42985 Experimental models: Cell lines 293T cells ATCC Cat# CRL-3216 Vero cells ATCC Cat# CRL-1586 ExpiCHO-S cells Thermo Fisher Scientific Cat# A29127 Experimental models: Organisms/strains K18-hACE2 Jackson Labs Cat# 034860 Recombinant DNA SARS-CoV-2 S2 Subunit with stabilizing disulfide mutations This study N/A Plasmid: 6C10 light chain This study N/A Plasmid: 6C10 heavy chain This study N/A Plasmid: 1A11 light chain This study N/A Plasmid: 1A11 heavy chain This study N/A Plasmid: 6E1 light chain This study N/A Plasmid: 6E1 heavy chain This study N/A Plasmid: 4B3 light chain This study N/A Plasmid: 4B3 heavy chain This study N/A Plasmid: 25.10C light chain Robinson et al. 56 N/A Plasmid: 25.10C heavy chain Robinson et al. 56 N/A (Continued on next page) 14 Cell Reports 44, 116052, August 26, 2025
Polymerase Chain Reaction:Article Title: Structure of a SARS-CoV-2 spike S2 subunit in a pre-fusion, open conformation.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Dulbecco’s Modified Eagle Medium High Glucose with Glutamax supplement Thermo Fisher Scientific Cat# 10566016 Dulbecco’s phosphate-buffered saline Corning Cat#21-031-CV N-hydroxysulfosuccinimide (Sulfo-NHS) ThermoFisher Scientific Cat# 24510 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Pierce Cat# PG82079 MES Carterra Cat# 3625 1M Ethanolamine, pH 8.5 Carterra Cat# 3638 10% Tween 20 Carterra Cat# 3631 0.2M Sodium Hydroxide Carterra Cat# 3638 Platinum SuperFi II Green PCR Master Mix Invitrogen Cat# 12369050 Platinum II Hot-Start Green PCR Master mix (2X) Thermo fisher Cat# 14001-013 NEBuilder HiFi DNA Assembly master mix NEB Cat# E2621L NEB 5 alpha F’Iq competent E coli NEB Cat# C2992I E-gel96 2% with SYBR safe Invitrogen Cat# G720802 Qiaprep 96 Turbo Miniprep kit Qiagen Cat# 27191 SARS-CoV-2-S2 subunit Spike protein ectodomains with VFLIP mutations and C-terminal Foldon, HRV3C protease cleavage site and twin-strep-tag This study N/A SARS-CoV-2 S-2P This Study N/A OptiPRO SFM Gibco Cat# 12309019 Critical commercial assays TMB substrate kit Peirce Cat# 34021 Plasmid Plus Midi ki Qiagen Cat# 12945 ExpiFectamine CHO Transfection kit ThermoFisher Cat# A29129 Deposited data 6C10 and VFLIP S2-DS5 complex (Cryo-EM) This study EMD-42985 Experimental models: Cell lines 293T cells ATCC Cat# CRL-3216 Vero cells ATCC Cat# CRL-1586 ExpiCHO-S cells Thermo Fisher Scientific Cat# A29127 Experimental models: Organisms/strains K18-hACE2 Jackson Labs Cat# 034860 Recombinant DNA SARS-CoV-2 S2 Subunit with stabilizing disulfide mutations This study N/A Plasmid: 6C10 light chain This study N/A Plasmid: 6C10 heavy chain This study N/A Plasmid: 1A11 light chain This study N/A Plasmid: 1A11 heavy chain This study N/A Plasmid: 6E1 light chain This study N/A Plasmid: 6E1 heavy chain This study N/A Plasmid: 4B3 light chain This study N/A Plasmid: 4B3 heavy chain This study N/A Plasmid: 25.10C light chain Robinson et al. 56 N/A Plasmid: 25.10C heavy chain Robinson et al. 56 N/A (Continued on next page) 14 Cell Reports 44, 116052, August 26, 2025
Plasmid Preparation:Article Title: Structure of a SARS-CoV-2 spike S2 subunit in a pre-fusion, open conformation.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Dulbecco’s Modified Eagle Medium High Glucose with Glutamax supplement Thermo Fisher Scientific Cat# 10566016 Dulbecco’s phosphate-buffered saline Corning Cat#21-031-CV N-hydroxysulfosuccinimide (Sulfo-NHS) ThermoFisher Scientific Cat# 24510 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Pierce Cat# PG82079 MES Carterra Cat# 3625 1M Ethanolamine, pH 8.5 Carterra Cat# 3638 10% Tween 20 Carterra Cat# 3631 0.2M Sodium Hydroxide Carterra Cat# 3638 Platinum SuperFi II Green PCR Master Mix Invitrogen Cat# 12369050 Platinum II Hot-Start Green PCR Master mix (2X) Thermo fisher Cat# 14001-013 NEBuilder HiFi DNA Assembly master mix NEB Cat# E2621L NEB 5 alpha F’Iq competent E coli NEB Cat# C2992I E-gel96 2% with SYBR safe Invitrogen Cat# G720802 Qiaprep 96 Turbo Miniprep kit Qiagen Cat# 27191 SARS-CoV-2-S2 subunit Spike protein ectodomains with VFLIP mutations and C-terminal Foldon, HRV3C protease cleavage site and twin-strep-tag This study N/A SARS-CoV-2 S-2P This Study N/A OptiPRO SFM Gibco Cat# 12309019 Critical commercial assays TMB substrate kit Peirce Cat# 34021 Plasmid Plus Midi ki Qiagen Cat# 12945 ExpiFectamine CHO Transfection kit ThermoFisher Cat# A29129 Deposited data 6C10 and VFLIP S2-DS5 complex (Cryo-EM) This study EMD-42985 Experimental models: Cell lines 293T cells ATCC Cat# CRL-3216 Vero cells ATCC Cat# CRL-1586 ExpiCHO-S cells Thermo Fisher Scientific Cat# A29127 Experimental models: Organisms/strains K18-hACE2 Jackson Labs Cat# 034860 Recombinant DNA SARS-CoV-2 S2 Subunit with stabilizing disulfide mutations This study N/A Plasmid: 6C10 light chain This study N/A Plasmid: 6C10 heavy chain This study N/A Plasmid: 1A11 light chain This study N/A Plasmid: 1A11 heavy chain This study N/A Plasmid: 6E1 light chain This study N/A Plasmid: 6E1 heavy chain This study N/A Plasmid: 4B3 light chain This study N/A Plasmid: 4B3 heavy chain This study N/A Plasmid: 25.10C light chain Robinson et al. 56 N/A Plasmid: 25.10C heavy chain Robinson et al. 56 N/A (Continued on next page) 14 Cell Reports 44, 116052, August 26, 2025
Transfection:Article Title: Structure of a SARS-CoV-2 spike S2 subunit in a pre-fusion, open conformation.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Dulbecco’s Modified Eagle Medium High Glucose with Glutamax supplement Thermo Fisher Scientific Cat# 10566016 Dulbecco’s phosphate-buffered saline Corning Cat#21-031-CV N-hydroxysulfosuccinimide (Sulfo-NHS) ThermoFisher Scientific Cat# 24510 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Pierce Cat# PG82079 MES Carterra Cat# 3625 1M Ethanolamine, pH 8.5 Carterra Cat# 3638 10% Tween 20 Carterra Cat# 3631 0.2M Sodium Hydroxide Carterra Cat# 3638 Platinum SuperFi II Green PCR Master Mix Invitrogen Cat# 12369050 Platinum II Hot-Start Green PCR Master mix (2X) Thermo fisher Cat# 14001-013 NEBuilder HiFi DNA Assembly master mix NEB Cat# E2621L NEB 5 alpha F’Iq competent E coli NEB Cat# C2992I E-gel96 2% with SYBR safe Invitrogen Cat# G720802 Qiaprep 96 Turbo Miniprep kit Qiagen Cat# 27191 SARS-CoV-2-S2 subunit Spike protein ectodomains with VFLIP mutations and C-terminal Foldon, HRV3C protease cleavage site and twin-strep-tag This study N/A SARS-CoV-2 S-2P This Study N/A OptiPRO SFM Gibco Cat# 12309019 Critical commercial assays TMB substrate kit Peirce Cat# 34021 Plasmid Plus Midi ki Qiagen Cat# 12945 ExpiFectamine CHO Transfection kit ThermoFisher Cat# A29129 Deposited data 6C10 and VFLIP S2-DS5 complex (Cryo-EM) This study EMD-42985 Experimental models: Cell lines 293T cells ATCC Cat# CRL-3216 Vero cells ATCC Cat# CRL-1586 ExpiCHO-S cells Thermo Fisher Scientific Cat# A29127 Experimental models: Organisms/strains K18-hACE2 Jackson Labs Cat# 034860 Recombinant DNA SARS-CoV-2 S2 Subunit with stabilizing disulfide mutations This study N/A Plasmid: 6C10 light chain This study N/A Plasmid: 6C10 heavy chain This study N/A Plasmid: 1A11 light chain This study N/A Plasmid: 1A11 heavy chain This study N/A Plasmid: 6E1 light chain This study N/A Plasmid: 6E1 heavy chain This study N/A Plasmid: 4B3 light chain This study N/A Plasmid: 4B3 heavy chain This study N/A Plasmid: 25.10C light chain Robinson et al. 56 N/A Plasmid: 25.10C heavy chain Robinson et al. 56 N/A (Continued on next page) 14 Cell Reports 44, 116052, August 26, 2025
Cryo-EM Sample Prep:Article Title: Structure of a SARS-CoV-2 spike S2 subunit in a pre-fusion, open conformation.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Dulbecco’s Modified Eagle Medium High Glucose with Glutamax supplement Thermo Fisher Scientific Cat# 10566016 Dulbecco’s phosphate-buffered saline Corning Cat#21-031-CV N-hydroxysulfosuccinimide (Sulfo-NHS) ThermoFisher Scientific Cat# 24510 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Pierce Cat# PG82079 MES Carterra Cat# 3625 1M Ethanolamine, pH 8.5 Carterra Cat# 3638 10% Tween 20 Carterra Cat# 3631 0.2M Sodium Hydroxide Carterra Cat# 3638 Platinum SuperFi II Green PCR Master Mix Invitrogen Cat# 12369050 Platinum II Hot-Start Green PCR Master mix (2X) Thermo fisher Cat# 14001-013 NEBuilder HiFi DNA Assembly master mix NEB Cat# E2621L NEB 5 alpha F’Iq competent E coli NEB Cat# C2992I E-gel96 2% with SYBR safe Invitrogen Cat# G720802 Qiaprep 96 Turbo Miniprep kit Qiagen Cat# 27191 SARS-CoV-2-S2 subunit Spike protein ectodomains with VFLIP mutations and C-terminal Foldon, HRV3C protease cleavage site and twin-strep-tag This study N/A SARS-CoV-2 S-2P This Study N/A OptiPRO SFM Gibco Cat# 12309019 Critical commercial assays TMB substrate kit Peirce Cat# 34021 Plasmid Plus Midi ki Qiagen Cat# 12945 ExpiFectamine CHO Transfection kit ThermoFisher Cat# A29129 Deposited data 6C10 and VFLIP S2-DS5 complex (Cryo-EM) This study EMD-42985 Experimental models: Cell lines 293T cells ATCC Cat# CRL-3216 Vero cells ATCC Cat# CRL-1586 ExpiCHO-S cells Thermo Fisher Scientific Cat# A29127 Experimental models: Organisms/strains K18-hACE2 Jackson Labs Cat# 034860 Recombinant DNA SARS-CoV-2 S2 Subunit with stabilizing disulfide mutations This study N/A Plasmid: 6C10 light chain This study N/A Plasmid: 6C10 heavy chain This study N/A Plasmid: 1A11 light chain This study N/A Plasmid: 1A11 heavy chain This study N/A Plasmid: 6E1 light chain This study N/A Plasmid: 6E1 heavy chain This study N/A Plasmid: 4B3 light chain This study N/A Plasmid: 4B3 heavy chain This study N/A Plasmid: 25.10C light chain Robinson et al. 56 N/A Plasmid: 25.10C heavy chain Robinson et al. 56 N/A (Continued on next page) 14 Cell Reports 44, 116052, August 26, 2025
Recombinant:Article Title: Structure of a SARS-CoV-2 spike S2 subunit in a pre-fusion, open conformation.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Dulbecco’s Modified Eagle Medium High Glucose with Glutamax supplement Thermo Fisher Scientific Cat# 10566016 Dulbecco’s phosphate-buffered saline Corning Cat#21-031-CV N-hydroxysulfosuccinimide (Sulfo-NHS) ThermoFisher Scientific Cat# 24510 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Pierce Cat# PG82079 MES Carterra Cat# 3625 1M Ethanolamine, pH 8.5 Carterra Cat# 3638 10% Tween 20 Carterra Cat# 3631 0.2M Sodium Hydroxide Carterra Cat# 3638 Platinum SuperFi II Green PCR Master Mix Invitrogen Cat# 12369050 Platinum II Hot-Start Green PCR Master mix (2X) Thermo fisher Cat# 14001-013 NEBuilder HiFi DNA Assembly master mix NEB Cat# E2621L NEB 5 alpha F’Iq competent E coli NEB Cat# C2992I E-gel96 2% with SYBR safe Invitrogen Cat# G720802 Qiaprep 96 Turbo Miniprep kit Qiagen Cat# 27191 SARS-CoV-2-S2 subunit Spike protein ectodomains with VFLIP mutations and C-terminal Foldon, HRV3C protease cleavage site and twin-strep-tag This study N/A SARS-CoV-2 S-2P This Study N/A OptiPRO SFM Gibco Cat# 12309019 Critical commercial assays TMB substrate kit Peirce Cat# 34021 Plasmid Plus Midi ki Qiagen Cat# 12945 ExpiFectamine CHO Transfection kit ThermoFisher Cat# A29129 Deposited data 6C10 and VFLIP S2-DS5 complex (Cryo-EM) This study EMD-42985 Experimental models: Cell lines 293T cells ATCC Cat# CRL-3216 Vero cells ATCC Cat# CRL-1586 ExpiCHO-S cells Thermo Fisher Scientific Cat# A29127 Experimental models: Organisms/strains K18-hACE2 Jackson Labs Cat# 034860 Recombinant DNA SARS-CoV-2 S2 Subunit with stabilizing disulfide mutations This study N/A Plasmid: 6C10 light chain This study N/A Plasmid: 6C10 heavy chain This study N/A Plasmid: 1A11 light chain This study N/A Plasmid: 1A11 heavy chain This study N/A Plasmid: 6E1 light chain This study N/A Plasmid: 6E1 heavy chain This study N/A Plasmid: 4B3 light chain This study N/A Plasmid: 4B3 heavy chain This study N/A Plasmid: 25.10C light chain Robinson et al. 56 N/A Plasmid: 25.10C heavy chain Robinson et al. 56 N/A (Continued on next page) 14 Cell Reports 44, 116052, August 26, 2025
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