key: cord-0786313-qme9btv2
authors: Cerutti, Gabriele; Guo, Yicheng; Liu, Lihong; Liu, Liyuan; Zhang, Zhening; Luo, Yang; Huang, Yiming; Wang, Harris H.; Ho, David D.; Sheng, Zizhang; Shapiro, Lawrence
title: Cryo-EM structure of the SARS-CoV-2 omicron spike
date: 2022-02-07
journal: Cell Rep
DOI: 10.1016/j.celrep.2022.110428
sha: b4a781509366d9a3863585ab3ad380070784468f
doc_id: 786313
cord_uid: qme9btv2

The recently reported B.1.1.529 omicron variant of SARS-CoV-2 includes 34 mutations in the spike protein relative to the Wuhan strain, including 15 mutations in the receptor binding domain (RBD). Functional studies have shown omicron to substantially escape the activity of many SARS-CoV-2-neutralizing antibodies. Here we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of the omicron spike protein ectodomain. The structure depicts a spike that is exclusively in the 1-RBD-up conformation with high mobility of RBD. Many mutations cause steric clashes and/or altered interactions at antibody binding surfaces, whereas others mediate changes of the spike structure in local regions to interfere with antibody recognition. Overall, the structure of the omicron spike reveals how mutations alter its conformation and explains its extraordinary ability to evade neutralizing antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a human pathogen 3 in 2019 in Wuhan, China, causing a disease now known as coronavirus disease 19 (COVID-19) 4 that is characterized by fever, acute respiratory illness, and pneumonia (Callaway et al., 2020; 5

Cucinotta and Vanelli, 2020; Zhou et al., 2020) . At the time of writing this article, more than 274 6 million infections had been reported worldwide, with over 5 million deaths 7 (https://arcg.is/0fHmTX, 2021). Numerous variants have been discovered through sequencing 8 over the past two years, with some major lineages designated as variants of concern (VOCs) due 9

to increased transmissibility, disease severity, resistance to neutralizing antibodies elicited by Here we present the cryo-EM structure of the omicron spike, which adopts an exclusively 1-up 50 RBD conformation. The overall architecture of the spike is conserved, but the mobility of RBDs 51 appears increased over other variants. Surface differences appear to be localized around sites of 52 antibody recognition, with serious implications for immune evasion. 53 54 Results 55

For structure determination, we produced a soluble version of the omicron spike corresponding to 57 residues 1 to 1208 of the ectodomain, and including two proline mutations in S2 which have been 58 previously used to stabilize the spike in its prefusion form (Walls et al., 2020; Wrapp et al., 2020) , 59 and a C-terminal His tag. The protein was expressed in Expi293 cells and purified by His-tag 60 affinity chromatography, and this protein was used for the preparation of cryo-EM grids. The 61 spike appeared to show a slight preferred orientation on the cryo-EM grids, so we collected data 62 J o u r n a l P r e -p r o o f with a 30 tilt angle. We collected and processed 13695 cryo-EM movies to obtain a 3D 63 reconstruction at 3.1 Å resolution ( Figure 1A, Figure S1 , Figure S2 , Table S1 ). 64

The overall structure is similar to the Wuhan spike, with only a 1-RBD up conformation identified 66 through ab initio map generation and 3D classification. The electron density for the RBD in the up 67 position is blurred compared to the density of the RBDs in the down position ( Figure 1A) . To 68 investigate this behavior we performed 3D variability analysis, a procedure that allows 69 visualization of structural heterogeneity, like partial occupancy and molecular motions, by P681H (proximal to the S1/S2 cleavage site) belonged to flexible regions that could not be resolved 85 in the cryo-EM structure. The remaining 30 mutations were visible in the cryo-EM map although 86 the side chains of mutated residues G142D, G339D, S477N, T478K and G496S were not resolved 87 ( Figure S2 , Table S2 ). The RBD mutations are mostly clustered near the inter-protomer RBD-88 RBD interface and many of them overlap with the ACE2 binding site, while the NTD mutations 89 are located in the flexible loops distal from the trimer axis. The S2 mutations are mostly located at 90 the top of the subunit, at the interface with S1. (Figure 2A) . The comparison revealed an overall root mean square 96 deviation (RMSD) of 1.1 Å and 0.6 Å for S1 and S2 subunits respectively. The measured distance 97 between NTD domains of the three protomers showed that the NTD domain from protomer A 98 (NTDA), which has an RBD up, is 5 Å closer to the NTD domain of protomer B (NTDB) than that 99 of the D614G spike ( Figure 2B) . We also observed that the S2 helix bundle (residues 988 to 1033) 100 has a shorter distance and increased buried accessible surface area (bASA) between protomers 101 than the wildtype spike ( Figure 2C and Table S3 ). 102

We then determined the center of mass (COM) for NTD, NTD', SD2, SD1, and RBD and used 104

COMs to calculate angles between these domains. The result revealed that protomer A has a 105 smaller angle between NTD', SD2, and SD1, while the angles between other domains are highly 106 similar to the wildtype spike ( Figure 2D ). The angles between the five domains in protomer B and 107 C have no difference compared to the wildtype. We then measured the bASA between the above 108 domains and found that almost every domain-domain interface bASA increases slightly in omicron 109 compared to the wildtype (Table S3) . Remarkably, NTD has a 3-fold increased bASA with 110 adjacent RBD, coupled to a 2 Å reduced distance between RBD and NTD through the orientation 111 change in NTD. In summary, our analyses showed increased inter-domain interactions of the 112 omicron spike. 113 114

We next mapped the omicron mutations to the spike structure and assessed their potential effects 116 on spike conformation. The majority of the RBD mutations are located in the receptor binding 117 motif (RBM), inner side, and outer side epitope regions ( Figure 3A ). The superimposition of the 118 omicron and wildtype RBDs showed an RMSD of 0.75 Å. We then calculated C distance for 119 each RBD residue between omicron and wildtype, and observed that six mutations (S371L, S373P, 120 S375F, G446S, S477N, and T478K) are located at regions with C distance larger than 2 Å 121 ( Figure 3B) , suggesting that these mutations may account for the conformational change in the 122 omicron RBD. In particular, we observed that mutations S371L, S373P, and S375F not only alter See also Figure S1 , Figure S2 , Figure S3 , Table S1 and Table S2 . and refinement statistics is shown in Table S1 . 394

The cryo-EM structural model and maps are in the process of being deposited in the RCSB PDB 395 and EMDB. 396 397

PyMOL was used to perform the angle and distance calculations and generate plots (Schrodinger, 399 2015) . We superposed different spikes by aligning the S2 domain in PyMOL. For each domain, 400

we selected a set of residues and used their C for determining the center of mass of the domain. 

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UCSF Chimera--a visualization system for exploratory research and analysis

UCSF ChimeraX: Structure visualization for researchers, educators, and 526 developers

Cross-neutralization of SARS-CoV-2 by a human monoclonal 529 SARS-CoV antibody

Sensitivity of infectious SARS-CoV-2 B.1.1.7 532 and B.1.351 variants to neutralizing antibodies

Considerable escape of SARS-CoV-2 Omicron 535 to antibody neutralization

3D variability analysis: Resolving continuous flexibility and 537 discrete heterogeneity from single particle cryo-EM

cryoSPARC: algorithms for 539 rapid unsupervised cryo-EM structure determination

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DeepEMhancer: a deep learning solution for cryo-EM volume post-processing

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RELION: implementation of a Bayesian approach to cryo-EM structure 550 determination

The PyMOL Molecular Graphics System

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Automated molecular microscopy: the new Leginon system

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Addressing preferred specimen orientation in single-particle cryo-EM through tilting

Improvement of 568 cryo-EM maps by density modification

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Overview of the CCP4 suite and current 589 developments

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Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody: mechanisms for 595 the high infectivity, immune evasion and antibody drug discovery

Structural and Functional Analysis of the D614G 598 SARS-CoV-2 Spike Protein Variant

Structural impact on SARS-CoV-2 spike protein by D614G 601 substitution

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Rapid isolation and profiling of a diverse panel of human 612 monoclonal antibodies targeting the SARS-CoV-2 spike protein