High-resolution electron microscopy revealed Omicron’s secrets


Several weeks ago, Lausanne became home to some of the world’s most powerful electron microscopes. They’re installed at the Dubochet Center for Imaging (DCI) – a joint research facility run by Ecole Polytechnique Federale de Lausanne (EPFL), the University of Lausanne and the University of Geneva – and could turn out to be a precious ally in the fight against SARS-CoV-2, and particularly the Omicron variant that’s spreading rapidly around the world.

Here we present a high-resolution structure of the Spike protein of Omicron, which is predicted to soon become the dominant SARS-CoV-2 variant worldwide (as of December 2021). The overall structure of the full-length Spike, its RBD and NTD domains are similar to that of the wild-type but with specific changes in both local conformations and surfaces involved in antibody recognition.

The location of the mutations in both the RBD and NTD suggests that there has been a strong evolutionary pressure on the Omicron Spike to evade the humoral immune response.

The high-quality maps in parts of the S2 domain allowed us to visualize Omicron-specific mutations that form new stabilizing interactions within the Spike trimer, and which may optimize its ability to engage and bind ACE2, while occluding epitopes that are targets of neutralizing antibodies.11

Even with a heavily mutated ACE2-binding interface, our binding experiments show that the Omicron RBD is capable of interacting with human ACE2 receptor as efficiently as that of the Delta variant. In addition, the robust binding of mouse ACE2 to the Omicron Spike suggests an increased ability to infect other animal species such as rodents.

Our structure highlights the many mutations found on the Omicron Spike, which we speculate may affect protein stability, glycan conformation diversity, membrane fusion, ACE2 and importantly evasion from antibodies from vaccination or previous infection.

During the completion of this manuscript a complementary preprint describing the Omicron Spike was released.31

Figure 1: Cryo-EM map and model of Spike Omicron Variant
a. Cryo-EM map of the Spike of the Omicron variant. Map coloring corresponds to each of the Spike monomer chains that comprise the full trimer (A (green), B (blue) and C (orange)). Red indicates a glycan. The flexibility of the single RBD-up (monomer C) is barely visible. b. Side- view surface representation of the atomic model of the Omicron Spike in gray, with mutations highlighted in yellow. c. Ribbon representation of monomer A, highlighting the different domains colored in gray-scale (as in panel d) and the mutations in spheres in yellow. Mutations are labelled. Red labelled mutations are those shared with other VOCs. d. Top view of panel b of the Omicron Spike, with specific mutations highlighted in yellow. e. Overview of the domain architecture of the Omicron Spike. Specific domains are highlighted: signal peptide (SP), N- terminal domain (NTD), receptor binding domain (RBD), S1 and S2 domain.
Figure 2: Omicron mutations alter local conformations and forms new interactions Zoomed in ribbon representations of the Omicron spike at sites of mutations. Mesh represents the cryo-EM map. Colors of models represent the Omicron Spike monomer chain as colored in Fig. 1a. a. D796Y mutation allows for the potential carbohydrate-pi interactions with N- linked glycans from N709 of a neighboring Spike monomer. b. N856K mutation of the fusion peptide. The longer side-chain of the lysine residue allows for a new interaction with T572 from an adjacent monomer. c. The N764K mutation forms a new network of interactions with both N317 and Q314 from an adjacent monomer. d. NTD mutation at a loop that contains a deletion at residue 211, a mutation L212I and an insertion of EPE at position 214. Numbering is based on that of the original wildtype Spike. e. Superposition of the same loop depicted in panel d. NTD-Omicron (dark blue) to the wild-type NTD (light blue) shows a large change in register and chemical environment of the loop between amino acids 210-215, wild-type numbering. f. Superposition of the RBD-Omicron (green) to the wild-type RBD (light green). Highlighted in the dashed-box is the receptor binding region that interacts with ACE2. Mutations present on the Omicron RBD are highlighted as orange spheres. g. Omicron RBD mutations represented at sticks (orange) centered on the mutations S371L, S373P, S375F. h. Omicron RBD mutations represented at sticks (orange) centered on the N501Y mutation.


The Omicron Spike cDNA was obtained by reverse transcription of a nasopharyngeal viral sample and cloned into an expression vector as previously described.3 The protein was purified to homogeneity via its TwinStrep tag from the supernatant of transiently transfected ExpiCHO cells (Fig. S1), and vitrified for cryo-electron microscopy (cryo-EM) at a concentration of 0.35 mg/ml as described in the Methods. Grids were screened with a 200kV TFS Glacios cryo-EM, and final data collection was performed on a 300kV TFS Titan Krios G4.

Data was processed ‘on-the-fly’ with CryoSPARC Live, resulting in a first 3D map after 3 hours from the Glacios.4 A total of 8672 dose-fractionated electron event recordings (movies) were then collected on the Titan Krios G4, processed first with cryoSPARC Live and re- processed manually with cryoSPARC version 3.3.1.

The resulting map of the full Omicron Spike was resolved at a resolution of 3.02 Å (FSC 0.143 criterion) with C1 symmetry (Fig. S2). A map with C3 symmetry was determined to a resolution of 2.65 Å. A local mask on a region containing both a receptor binding domain (RBD) and a N-terminal domain (NTD) was applied. Focused refinement specifically on this masked region yielded a map at 3.88 Å resolution.

The cryo-EM maps of the Omicron Spike glycoprotein clearly revealed many of the mutations present in this variant (Fig. S3). The overall architecture of the Spike trimer was comparable to the first structure of the wild-type Spike (PDB ID 6VSB) (RMSD Cɑ all atoms 2.4 Å) with one RBD in the up position and two RBDs down (Fig. 1a-b)5, although a minority of Spike trimers were in a closed conformation with all RBDs down.

We did not observe other populations containing two or three RBDs in the up configuration. This suggests that the Omicron Spike may preferentially be in either a one RBD-up or in a closed state (Fig. S2). The best-defined mutations are within the S2 domain intercalating between Spike monomers and within the NTD (Fig. 1c, e). A view from the top of the Spike protein exposes a large cluster of mutations on the RBDs (Fig. 1d-e).

The exceptional quality of the map in the S2 domain reveals potential new interactions formed by Omicron-unique mutations. The D796Y (all numbering based on wild-type positions) mutation replaces a charged surface-exposed acidic residue with tyrosine, an amino acid that contains an aromatic sidechain (Fig. 2a). We observe that Y796 allows for potential carbohydrate-pi interactions with the N-linked glycan chain originating from N709 of the neighboring monomer chain.6,7 We suspect that this interaction could have a stabilizing effect for the spike trimer.

The N856K mutation is within the fusion peptide of the Spike protein.8 The fusion peptide is required for attachment and anchoring of the virus directly to a host’s cell membrane. It is exposed during proteolytic cleavage that liberates the S1 from the S2 domain followed by subsequent cleavage at the S2’ site. We observe the N856K mutation with the longer side-chain of the lysine residue able to form new interactions with T572 from an adjacent monomer (Fig. 2b). We can only speculate that this may have a functional impact on the release of the fusion peptide. In addition, the mutation from an asparagine to lysine may aid cell membrane penetration.8–10

Other mutations located within S2 with stabilizing potential, such as N764K, can also be clearly visualized (Fig. 2c). We can model an amine head-group of K764 forming two new interactions with Q314 and N317 from an adjacent monomer, which could stabilize the Spike trimer. In particular, Q314 and N317 precede the RBD in sequence (residues 330-520) and are just below the linker that allows for RBD transition from a down to an up position.

We observe that the RBD is predominantly in a 1-up or closed state, allowing for the Spike to occlude efficiently RBD epitopes. Thus, the many mutations present in the S2 domain present only in the Omicron Spike protein may allow for optimization of RBD positions that allow for receptor binding while concealing epitopes that are the targets of neutralizing antibodies.11

The NTD is the target of various neutralizing antibodies and constitutes an antigenic supersite,12–14 and was subjected to focused refinement (Fig. 2d-h). We observe that the overall fold of the Omicron NTD is relatively unchanged compared to either its wild-type (RMSD 1.7 Å) or Delta (RMSD 2.4 Å) counterparts (Fig. S4a-b). The loop encompassing deletions 143- 145 (N3 loop)12–14 has a different conformation compared to the Delta NTD. The Delta variant Spike also contains NTD mutations in the same N3 loop (E156G, Δ157-158).

Superpositions of structures of Fab-bound NTDs predict that the Omicron NTD is resistant to some NTD antibodies due to steric clashes and changes in the antibody binding surface (Fig. S4c).12–14 We identify a second loop (residues 208-217), hereby called the EPE loop, which contains Omicron-specific mutations, deletions and inserts: deletion 211, L212I and insertion at 214 of EPE (Fig2. d-e).

The locally-refined cryo-EM map in this region allowed for the de novo building of this loop with the aid of a model generated from AF2.15 In the Omicron NTD, the EPE loop follows the Cɑ backbone as the wild-type NTD, however the chemical nature is drastically changed with the introduction of two acidic negatively charged residues (Fig. 2e). This insertion is not predicted to affect the binding of known NTD targeting antibodies, being localized far from the site of antibody binding (Fig. S4c).12,13

The RBD is the target for a vast number of neutralizing antibodies elicited by vaccination and previous infection. All commercially available antibodies that have been approved for clinical use, target this domain of the Spike protein. Based upon previous variants of concern (VOCs) and experimental studies, some of the mutations such as N501Y and Q498R may independently or synergistically enhance binding of the Spike to its receptor, the human ACE2 protein.16,17

In addition, the mutations that decorate the RBD may allow for immune evasion or render monoclonal antibody therapy ineffective.18–20 Alignment of the wild-type RBD with that of the Omicron reveals only slight changes with a RMSD of 2.6 Å (Fig. 2f). The locally-refined map in this region allowed us to model all the mutations located within the RBD (Fig. 2g-h and S3). A region that encompasses residues 371 to 375 contains three serine residues mutated: S371L, S373P, S375F.

These residues are on a surface exposed region of the RBD and may play a role in immune evasion of the Omicron variant. In addition, the top saddle of the RBD (Fig. 2f) is the location of many mutations shared with others VOCs, in particular N501Y that has been shown to foster ACE2 binding, along with other Omicron-specific mutations such as Q493R and Q498R.16,17

Overall the Omicron RBD has a highly mutated surface that encompasses the epitopes of the known major classes of antibodies (Fig. S5a). Superpositions of structures of representative Fab-bound RBDs suggest that all 4 classes of RBD targeting antibodies have their ability to bind abrogated (Fig. S5b).21–27 These observations suggest that the Omicron variant has evolved immune evading mutations from various classes of RBD-targeting antibodies.

Many of the RBD mutations are also located along a ridge that interacts directly with

the helices of the ACE2 receptor (Fig. 2f). We performed biolayer interferometry (BLI) binding assays to evaluate the affinity of ExpiCHO-expressed dimeric Fc-human ACE2 to full-length Omicron Spike (Fig. S6). Compared to the wild-type Spike, hACE2 binds to the Omicron Spike with similar affinities and comparable to that of binding to the Delta Spike (4.4 nM vs. 6.8 nM vs. 6.1 nM; wild-type vs. Omicron vs. Delta).

The Omicron Spike also shares many RBD- specific mutations (namely N501Y, K417N and mutation at E484) with other VOCs, namely Alpha, Beta and Gamma. These variants all show relatively stronger binding to hACE2 than Omicron (Fig. S6). We conclude that the appearance of a large number of mutations at the ACE2 binding interface do not significantly alter the overall binding affinity of dimeric Fc- hACE2 to the Omicron Spike.

In addition, we tested binding to the dimeric mouse ACE2 receptor as it has been previously reported that N501Y containing VOCs had increased ability to infect mice and bind ACE2.28–30 The dimeric Fc-mACE2 displays robust binding to the Omicron Spike with an almost 8-fold increase in binding affinity compared to wild-type.

Mouse ACE2 also binds significantly stronger compared to other variants with the exception of gamma.

reference link : Dongchun Ni et al, Structural analysis of the Spike of the Omicron SARS-COV-2 variant by Cryo-EM and implications for immune evasion, (2021). DOI: 10.1101/2021.12.27.474250


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