Journal: Cell reports

Article Title: Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein.

doi: 10.1016/j.celrep.2022.111964

Figure Lengend Snippet: Figure 2. Binding affinity and cryo-EM structure of the Omicron BA.2 S protein-human ACE2 complex (A) Surface plasmon resonance experiments measuring dimeric human ACE2 (hACE2) binding to immobilized wild-type (WT), BA.1, and BA.2 RBDs, performed in technical triplicates. Summary data are shown at the top with representative surface plasmon resonance (SPR)-binding curves (colored solid line), and fitted 1:1 binding models (black dashed line) are shown on bottom. (B) As in (A) but measuring WT, BA.1, and BA.2 RBDs binding to immobilized dimeric hACE2, performed in at least technical quadruplicates. (C) As in (A) but measuring WT, BA.1, and BA.2 ectodomains binding to immobilized dimeric hACE2, performed in at least technical duplicates. The WT and BA.1 data in (C) were previously reported.9 Pairwise statistical significance test was performed using a one-way ANOVA test (*p % 0.05; **p % 0.01; ***p % 0.001; ****p % 0.0001, ns, not significant). (D) Focus-refined cryo-EM density map and fitted atomic model of the BA.2 RBD in complex with hACE2 at 2.8 A˚ . (E) Aligned atomic models of hACE2 bound to BA.1 and BA.2 RBDs. The BA.1 RBD (PDB: 7T9L) and complexed hACE2 atomic models are shown in magenta and dark blue, respectively. The BA.2 RBD and complexed hACE2 atomic models are shown in purple and light blue, respectively. (F) Atomic model of the BA.1 S protein-hACE2 complex, focused on residue S496. The hydrogen bonding interaction between BA.1 S protein residue S496 and hACE2 residue K353 is indicated by an orange dashed line. (G) As in (F) but for the BA.2 S protein-hACE2 complex, focused on residue G496.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies VH-FC ab8 (Li et al.)18 N/A Fab S309 (Pinto et al.)20 N/A Fab S2M11 (Tortorici et al.)19 N/A Fab 4-8 (Liu et al.)21 N/A Fab 4A8 (Chi et al.)22 N/A Fab DH1052 (Li et al.)23 N/A Goat anti-human IgG Jackson ImmunoReserach Cat. # 109-035-088; RRID: AB_2337584 Mouse anti-his tag antibody abcam Cat# ab18184; RRID: AB_444306 Chemicals, peptides, and recombinant proteins Ace2 (18-615) New England Biolabs Cat. # 73775S BA.1 RBD Sino Biological Cat# 40592-V08H121 Critical commercial assays Pierce 1-Step Ultra Substrate Solution ThermoFisher Cat. # 34028 Deposited data S(BA.2) This paper Global refinement: EMDB 27523, PDB: 8DM1 Focus refinement: EMDB 27524, PDB: 8DM2 S(BA.2)+hACE2 This paper Global refinement: EMDB 27527, PDB: 8DM5 Focus refinement: EMDB 27528, PDB: 8DM6 S(BA.2)+mACE2 This paper Global refinement: EMDB 27529, PDB: 8DM7 Focus refinement: EMDB 27530, PDB: 8DM8 S(BA.1)+mACE2 This paper Global refinement: EMDB 27531, PDB: 8DM9 Focus refinement: EMDB 27532, PDB: 8DMA S(BA.2)+4A8 This paper Global refinement: EMDB 27525, PDB: 8DM3 Focus refinement: EMDB 27526, PDB: 8DM4 Experimental models: Cell lines Expi293F ThermoFisher Cat# A14527 Recombinant DNA pcDNA3.1 HexaPro BA.1 Mannar et al.9 N/A pcDNA3.1 BA.1 NTD This paper N/A pcDNA3.1 HexaPro BA.2 This paper N/A pcDNA3.1 BA.2 NTD This paper N/A pcDNA3.1 BA.2 RBD This paper N/A pcDNA3.1 hACE2 (1-615) Mannar et al.9 N/A pcDNA3.1 mACE2 (1-615) This paper N/A pCSCG hACE2-FC Yang et al.39 Addgene: 164222 pcDNA3.1 Fab S309 Light Chain Mannar et al.30 N/A pcDNA3.1 Fab S309 Heavy Chain Mannar et al.30 N/A pcDNA3.1 Fab S2M11 Light Chain Mannar et al.30 N/A pcDNA3.1 Fab S2M11 Heavy Chain Mannar et al.30 N/A pcDNA3.1 Fab 4-8 Light Chain Saville et al.40 N/A pcDNA3.1 Fab 4-8 Heavy Chain Saville et al.40 N/A pcDNA3.1 Fab 4A8 Light Chain Saville et al.40 N/A pcDNA3.1 Fab 4A8 Heavy Chain Saville et al.40 N/A pcDNA3.1 Fab DH1052 Light Chain This paper N/A (Continued on next page) 10 Cell Reports 42, 111964, January 31, 2023

Techniques: Binding Assay, Cryo-EM Sample Prep, SPR Assay, Residue

Journal: Cell reports

Article Title: Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein.

doi: 10.1016/j.celrep.2022.111964

Figure Lengend Snippet: Figure 3. Cryo-EM structure of the Omicron BA.2 S protein-mouse ACE2 complex (A) Cryo-EM density map of BA.2 S protein in complex with mouse ACE2 at 2.5 A˚ . Mouse ACE2 is shown in green, and protomers of the BA.2 S protein are shown in shades of purple. (B) Focus-refined cryo-EM density map and fitted atomic model of the BA.2 RBD-mouse ACE2 (mACE2) complex at 2.7 A˚ . (C) Aligned atomic models of the BA.1 and BA.2 RBD-mACE2 complexes. The BA.1 RBD and complexed hACE2 atomic models are shown in magenta and dark green, respectively. The BA.2 RBD and complexed hACE2 atomic models are shown in purple and light green, respectively. (D) Atomic model of the BA.2 RBD-mACE2 complex, focused on residues Y501 and H505. (E) As in (D) but focused on residue R493. (F) Atomic model of the WT RBD-hACE2, focused on residues N501 and Y505. (G) As in (H) but focused on residue Q493. (H) Atomic model of mACE2 from the perspective of a binding RBD. Black labels are mACE2 residues, and gray labels denote the interacting residues in a bound RBD. Gold labels denote the interacting residues in a bound RBD that are mutated in the BA.1 and BA.2 Omicron sub-lineages.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies VH-FC ab8 (Li et al.)18 N/A Fab S309 (Pinto et al.)20 N/A Fab S2M11 (Tortorici et al.)19 N/A Fab 4-8 (Liu et al.)21 N/A Fab 4A8 (Chi et al.)22 N/A Fab DH1052 (Li et al.)23 N/A Goat anti-human IgG Jackson ImmunoReserach Cat. # 109-035-088; RRID: AB_2337584 Mouse anti-his tag antibody abcam Cat# ab18184; RRID: AB_444306 Chemicals, peptides, and recombinant proteins Ace2 (18-615) New England Biolabs Cat. # 73775S BA.1 RBD Sino Biological Cat# 40592-V08H121 Critical commercial assays Pierce 1-Step Ultra Substrate Solution ThermoFisher Cat. # 34028 Deposited data S(BA.2) This paper Global refinement: EMDB 27523, PDB: 8DM1 Focus refinement: EMDB 27524, PDB: 8DM2 S(BA.2)+hACE2 This paper Global refinement: EMDB 27527, PDB: 8DM5 Focus refinement: EMDB 27528, PDB: 8DM6 S(BA.2)+mACE2 This paper Global refinement: EMDB 27529, PDB: 8DM7 Focus refinement: EMDB 27530, PDB: 8DM8 S(BA.1)+mACE2 This paper Global refinement: EMDB 27531, PDB: 8DM9 Focus refinement: EMDB 27532, PDB: 8DMA S(BA.2)+4A8 This paper Global refinement: EMDB 27525, PDB: 8DM3 Focus refinement: EMDB 27526, PDB: 8DM4 Experimental models: Cell lines Expi293F ThermoFisher Cat# A14527 Recombinant DNA pcDNA3.1 HexaPro BA.1 Mannar et al.9 N/A pcDNA3.1 BA.1 NTD This paper N/A pcDNA3.1 HexaPro BA.2 This paper N/A pcDNA3.1 BA.2 NTD This paper N/A pcDNA3.1 BA.2 RBD This paper N/A pcDNA3.1 hACE2 (1-615) Mannar et al.9 N/A pcDNA3.1 mACE2 (1-615) This paper N/A pCSCG hACE2-FC Yang et al.39 Addgene: 164222 pcDNA3.1 Fab S309 Light Chain Mannar et al.30 N/A pcDNA3.1 Fab S309 Heavy Chain Mannar et al.30 N/A pcDNA3.1 Fab S2M11 Light Chain Mannar et al.30 N/A pcDNA3.1 Fab S2M11 Heavy Chain Mannar et al.30 N/A pcDNA3.1 Fab 4-8 Light Chain Saville et al.40 N/A pcDNA3.1 Fab 4-8 Heavy Chain Saville et al.40 N/A pcDNA3.1 Fab 4A8 Light Chain Saville et al.40 N/A pcDNA3.1 Fab 4A8 Heavy Chain Saville et al.40 N/A pcDNA3.1 Fab DH1052 Light Chain This paper N/A (Continued on next page) 10 Cell Reports 42, 111964, January 31, 2023

Techniques: Cryo-EM Sample Prep, Residue, Binding Assay