key: cord-0946094-ofht7g1p
authors: Wu, Nicholas C.; Yuan, Meng; Bangaru, Sandhya; Huang, Deli; Zhu, Xueyong; Lee, Chang-Chun D.; Turner, Hannah L.; Peng, Linghang; Yang, Linlin; Burton, Dennis R.; Nemazee, David; Ward, Andrew B.; Wilson, Ian A.
title: A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody
date: 2020-12-04
journal: PLoS Pathog
DOI: 10.1371/journal.ppat.1009089
sha: 66dc15dc3583ddff0a9a9977ba8ef3a393d48919
doc_id: 946094
cord_uid: ofht7g1p

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

While CR3022 can neutralize SARS-CoV [18, 20] , multiple groups have shown that it does not neutralize SARS-CoV-2 [3, 5, 20, 22] . One possibility is that the affinity of CR3022 to SARS-CoV-2 RBD is not sufficient to confer neutralizing activity. To test this hypothesis, we compared neutralization of SARS-CoV-2 WT and the P384A mutant by CR3022. Consistent with previous studies [3, 5, 20, 22] , CR3022 failed to neutralize SARS-CoV-2 WT (Fig 2) . However, CR3022 is now able to neutralize the SARS-CoV-2 P384A mutant at an IC 50 of 3.2 μg/ml, which is comparable to its neutralizing activity to SARS-CoV (IC 50 of 5.2 μg/ml). This finding validates the CR3022 epitope as a neutralizing epitope in both SARS-CoV-2 and SARS-CoV, provided that the antibody affinity can surpass a threshold for detection of neutralization.

Previous studies have indicated IgG bivalent binding can play an important role in mediating neutralization of SARS-CoV-2, since the neutralization potency for many antibodies is much greater as an IgG compared to an Fab [21, 23] . Subsequently, we also tested the neutralizing activity of CR3022 Fab. Interestingly, the CR3022 Fab neutralized SARS-CoV-2 P384A mutant with an IC 50 of 4.4 μg/ml, which is similar to that of CR3022 IgG (3.2 μg/ml) (Fig 2) . This finding indicates that CR3022, unlike many other SARS-CoV-2 antibodies [21, 23] , does not act bivalently with the S proteins on the virus surface and, hence, neutralization is related to the Fab binding affinity rather than IgG avidity.

We then examined the sequence conservation of residue 384 in other SARS-related coronaviruses (SARSr-CoV) strains. Most SARSr-CoV strains have Pro at residue 384, as in SARS- 

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody CoV-2. Only those strains that are phylogenetically very close to SARS-CoV, such as bat SARSr-CoV WIV1 and bat SARSr-CoV WIV16, have Ala at residue 384 (Figs 3A and S1). Phylogenetic analysis implies that P384A emerged during the evolution of SARSr-CoV in bats (Figs 3A and S1), which is the natural reservoir of SARSr-CoV [24] . However, it is unclear whether the emergence of P384A is due to neutral drift or positive selection in bats or other species. In addition, given that residue 384 is proximal to the S2 domain when the RBD is in the "down" conformation ( Fig 3B) , whether P384A can modulate the conformational dynamics of the "up and down" configurations of the RBD in the S trimer and influence the viral replication fitness will require additional studies.

We further determined the x-ray structure of SARS-CoV RBD in complex with CR3022 to 2.7 Å resolution (Figs 4A and S2 and S1 Table) . The overall structure of CR3022 in complex with SARS-CoV RBD is similar to that with SARS-CoV-2 RBD [20] (Cα RMSD of 0.5 Å for 343 residues in the RBD and Fab variable domain, cf. Fig S2A and S2B of [20] ) (S3 Fig). Nonetheless, the CR3022 elbow angles, which are distant from the antibody-antigen interface, differ in the two structures, as we mutated the elbow region (as described in [25] ) of CR3022 to promote crystallization with SARS-CoV RBD. The conserved binding mode of CR3022 to SARS-CoV-2 RBD and SARS-CoV RBD indicates that the difference in binding affinity of CR3022 between SARS-CoV-2 RBD and SARS-CoV RBD is therefore due only to a very subtle structural difference.

To investigate how P384 and A384 lead to differential binding of CR3022, we compared RBD structures from SARS-CoV and SARS-CoV-2 when bound to CR3022. The RBDs have a Cα RMSD of only 0.6 Å (0.7 Å for CR3022 epitope residues). At residue 384, the backbone of SARS-CoV-2 is further from CR3022, as compared to that of SARS-CoV ( Fig 4B) . This difference in backbone positioning (~1.3 Å shift) affects the interaction of the RBD with CR3022 V H S96, which is encoded by IGHD3-10 gene segment on CDR H3 [18, 20] . While CR3022 V H S96 forms a hydrogen bond (H-bond) with the T385 side chain in both SARS-CoV-2 RBD and SARS-CoV RBD, it can form a second H-bond with the backbone amide of T385 in [31] ). S1 domain is represented by the white surface and the S2 domain by the black cartoon. The location of residue 384 is indicated by the red sphere on the RBD in the "down" conformation (blue cartoon). CR3022 is not shown in this figure.

https://doi.org/10.1371/journal.ppat.1009089.g003

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody SARS-CoV RBD (Fig 4C) , but not SARS-CoV-2 RBD (Fig 4D) . In addition, CR3022 V H S96 adopts different side-chain rotamers when binding to SARS-CoV-2 and to SARS-CoV. Consequently, V H S96 can make an intramolecular H-bond with V H T31 when CR3022 binds to SARS-CoV RBD (Fig 4C) , but not to SARS-CoV-2 ( Fig 4D) . In summary, V H S96 forms three H-bonds when CR3022 binds to SARS-CoV RBD, as compared to only one when CR3022 binds to SARS-CoV-2 RBD. This observation indicates why binding of CR3022 to the SARS--CoV RBD is energetically more favorable than to the SARS-CoV-2 RBD.

To understand the binding of CR3022 to the RBD in the context of the homotrimeric S protein, we previously proposed a structural model where CR3022 could only access its epitope on the S protein when at least two RBD are in the "up" conformation and the RBD is rotated relative to its unliganded structure [20] . To further evaluate and expand on this model, negative-stain electron microscopy (nsEM) was performed on CR3022 in complex with a stabilized version of the SARS-CoV homotrimeric S protein (Fig 5A, see Materials and Methods). The 3D nsEM reconstruction revealed that one SARS-CoV S protein could simultaneously bind to three CR3022 Fabs with all three RBDs in the "up" conformation ( Fig 5B) . Consistent with the structural model that we previously proposed [20] , the CR3022-bound RBD was indeed rotated compared to that in the unliganded S protein [26] [27] [28] , such that, in this conformation, steric hinderance between CR3022 and the N-terminal domain (NTD) is minimized.

While our results here demonstrate that CR3022 Fab could form a stable complex with SARS-CoV S protein in a prefusion conformation, a recent study reported that prefusion SARS-CoV-2 S protein fell apart upon binding to CR3022 Fab as indicated by cryo-EM [29] . It should be noted that the three-up conformation is much more rarely observed than the other RBD conformations (all-down, one-up, and two-up) in SARS-CoV by cryo-EM [26] [27] [28] , or SARS-CoV-2 by cryo-EM [30] [31] [32] and cryo-electron tomography [33, 34] , and could relate to differences in the stability of S trimers in SARS-CoV versus SARS-CoV-2 when CR3022 is bound. Further studies will be required to investigate whether such a difference between SARS-CoV-2 and SARS-CoV is related to stability differences in the recombinant spike proteins, or to different dynamics of the RBD on the virus or infected cells. 

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

To address some of these issues, we performed cryo-EM analysis to interrogate the binding of CR3022 to SARS-CoV S protein at higher resolution (S4 Fig and S2 Table) . Focused 3D [35] is also fit into density with one RBD removed for clarity. The protomers are colored in purple, magenta and deep magenta. (D) Top view of the class 2 cryo-EM map depicting potential quaternary contacts between the RBD-bound Fab and the spike NTD in this conformation. In this RBD-Fab conformation, the Fab would clash with the "down" RBD of the adjacent protomer (magenta) and, therefore, the adjacent RBD can only exist in an "up" conformation. (E) A close-up view of the Fab-spike interface showing the superimposition of CR3022 Fab and adjacent RBD. The residues that can contribute to quaternary interactions between CR3022 light chain and the NTD in two of the four classes (2 and 4) are shown.

https://doi.org/10.1371/journal.ppat.1009089.g005

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody classification yielded 4 different structural classes with classes 2 and 4 being nearly identical at the given resolution (Figs 5C and S5). Class 3 is the most similar to the model from nsEM, although the total particle number for classes 2 and 4 together exceed that for class 3 (S5 Fig). In contrast, class 1 is the least represented. In classes 2 and 4, CR3022 also appears to make quaternary contacts with the NTD, as suggested by well-defined density in the CR3022-NTD interface (Fig 5C) . The moderate resolution (6 to 7 Å) of the reconstructions precludes atomic-level descriptions, but the framework region of the CR3022 light chain in classes 2 and 4 is in close proximity to a loop region in NTD corresponding to residues 106-110. In addition, the constant region of CR3022 appears to contact residue D23 of NTD. Another notable observation is that the Fab in class 2 and 4 would clash with the adjacent RBD if it were in the "down" conformation. So, for the Fab to exist in this quaternary conformation, the adjacent RBD has to be in the "up" conformation. To evaluate the different dispositions of the RBD in these structures, we compared the cryo-EM structure of an apo form of the SARS-CoV S protein where one RBD is the "up" conformation (PDB 6ACD) [35] . Overall, these structural analyses indicate that RBD rotational flexibility and acquisition of quaternary interactions can play an important role in CR3022 interaction with the S protein. CR3022 adds to the growing list of neutralization antibodies that can utilize quaternary interactions for binding to the S protein [12, 36] .

Despite the flexibility of CR3022-bound RBD, bivalent binding of CR3022 to S protein does not seem to occur on the virus surface since an IgG avidity effect was not observed in the neutralization assay (see above, Fig 2) .

While it is now known that SARS-CoV and SARS-CoV-2 differ in antigenicity despite relatively high sequence conservation [1, 3, 4, 14] , there is a paucity of understanding of the underlying molecular determinants of these antigenic changes and the structural consequences of these differences. Through structural analysis of the CR3022-RBD complex and mutagenesis experiments, we show that a single amino-acid substitution at residue 384 contributes to an important antigenic difference in a highly conserved (neutralizing) epitope between SARS--CoV-2 and SARS-CoV.

While CR3022 cannot neutralize SARS-CoV-2 WT in almost all studies [3, 5, 20, 22] , it can neutralize the SARS-CoV-2 P384A mutant. The K D of CR3022 Fab to SARS-CoV-2 WT RBD is 68 nM, whereas to SARS-CoV-2 P384A RBD is 1 nM (Fig 1B and 1C) , indicating that the affinity threshold for neutralization of SARS-CoV-2 to this epitope is in the low nM range. However, despite having a low nM affinity to SARS-CoV-2 P384A RBD, CR3022 only weakly neutralizes SARS-CoV-2 P384A with an IC 50 of 3.2 μg/ml and SARS-CoV with an IC 50 of 5.2 μg/ml. In contrast, antibodies with similar or less Fab binding affinity to other RBD epitopes, such as the receptor binding motif, can neutralize SARS-CoV-2 much more efficiently. For example, previously characterized SARS-CoV-2 antibodies CC12.1 and CC12.3, which

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody have a K D of 17 nM and 14 nM to SARS-CoV-2 RBD, respectively, neutralize SARS-CoV-2 at an IC 50 of~20 ng/ml [3, 37] . Of note, the K D and IC 50 of CC12.1 and CC12.3 were measured in the same manner as this study. The lack of correlation between affinity and neutralizing activity is therefore not due to the difference in the assays between studies. In fact, a previous study also demonstrated a lack of correlation between RBD binding and neutralization for monoclonal antibodies [3] . Together, these observations suggest that the affinity threshold for SARS--CoV-2 neutralization is different for different RBD-targeting antibodies.

The difference in affinity threshold for different epitopes is likely to be related not only in the ability to block ACE2-binding [3, 38] , but also in antibody avidity where bivalent binding can cross-link different RBD domains on the same or different spikes and, hence, substantially enhance binding and neutralization [23, 39] . Since we first reported the structure of CR3022 in complex with SARS-CoV-2 RBD [20] , multiple cross-neutralizing antibodies, including COVA1-16 [39] , EY6A [17] , H014 [40] , and ADI-56046 [6] , have been shown to bind epitopes that largely overlap with the CR3022 epitope. One of these antibodies, COVA1-16, has a strong IgG avidity effect in the neutralization assay in contrast to CR3022 [39] . Such a drastic difference in IgG avidity between CR3022 and COVA1-16 may be due to their very different angles of approach in binding to RBD, which may in turn accommodate bivalent binding of IgG COVA1-16 but not CR3022 (S8 Fig). As IgG avidity continues to emerge as an explanation for the observed potency of SARS-CoV-2 neutralizing antibodies often with little to no somatic mutations [41] , future studies should investigate which epitopes and antibody approach angles give rise to avidity to the spike protein on the virus.

Given the scale of the outbreak, SARS-CoV-2 may persist and circulate in humans for years to come [42] . A number of SARS-CoV-2 vaccine candidates are currently under clinical trials (https://clinicaltrials.gov/ct2/who_table) [43] , which offer a potential solution to alleviate the global health and socio-economic devastation bought by SARS-CoV-2. However, whether SARS-CoV-2 can escape vaccine-induced immunity through antigenic drift remains to be determined, although escape mutations to many monoclonal antibodies have been tested in vitro [2] . Identification of the key residues that are responsible for differences in antigenicity among SARS-CoV-2, SARS-CoV, and possibly other SARS-related viruses, should provide a starting point to understand the potential for antigenic drift in SARS-like coronaviruses. The ongoing efforts in SARS-CoV-2 antibody discovery and structural characterization will therefore advance our molecular understanding of antigenic variation in SARS-like CoVs, and consequences for vaccine and therapeutic design, especially to cross-neutralizing epitopes, which could aid in protection against future epidemics or pandemics.

RBD (residues 319-541) of the SARS-CoV-2 spike protein (GenBank: QHD43416.1) and RBD (residues: 306-527) of the SARS-CoV spike (S) protein (GenBank: ABF65836.1) were fused with an N-terminal gp67 signal peptide and a C-terminal His 6 tag, and cloned into a customized pFastBac vector [44] . Recombinant bacmid DNA was generated using the Bac-to-Bac system (Thermo Fisher Scientific). Baculovirus was generated by transfecting purified bacmid DNA into Sf9 cells using FuGENE HD (Promega), and subsequently used to infect suspension cultures of High Five cells (Thermo Fisher Scientific) at an MOI of 5 to 10. Infected High Five cells were incubated at 28˚C with shaking at 110 r.p.m. for 72 h for protein expression. The supernatant was then concentrated using a 10 kDa MW cutoff Centramate cassette (Pall Corporation). For the binding study, constructs were cloned into phCMV3 and transiently transfected into Expi293F cells using ExpiFectamine 293 Reagent (Thermo Fisher Scientific)

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody according to the manufacturer's instructions. The supernatant was collected at 7 days posttransfection. The RBD proteins were purified by Ni-NTA, followed by size exclusion chromatography, and buffer exchanged into 20 mM Tris-HCl pH 7.4 and 150 mM NaCl.

The SARS-CoV spike construct (Tor2 strain) for recombinant spike protein expression contains the mammalian-codon-optimized gene encoding residues 1-1190 of the spike followed by a C-terminal T4 fibritin trimerization domain, a HRV3C cleavage site, 8x-His tag and a Twin-strep tags subcloned into the eukaryotic-expression vector pαH. Residues at 968 and 969 were replaced by prolines for generating stable spike proteins as described previously [28] . The spike plasmid was transfected into FreeStyle 293F cells and cultures were harvested at 6-day post-transfection. Proteins were purified from the supernatants on His-Complete columns using a 250 mM imidazole elution buffer. The elution was buffer exchanged to Tris-NaCl buffer (25 mM Tris, 500 mM NaCl, pH 7.4) before further purification using Superose 6 increase 10/300 column (GE Healthcare). Protein fractions corresponding to the trimeric spike proteins were collected and concentrated.

The CR3022 Fab heavy (GenBank: DQ168569.1) and light (GenBank: DQ168570.1) chains were cloned into phCMV3. The plasmids were transiently co-transfected into Expi293F cells at a ratio of 2:1 (HC:LC) using ExpiFectamine 293 Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The supernatant was collected at 7 days post-transfection. The Fab was purified with a CaptureSelect CH1-XL Pre-packed Column (Thermo Fisher Scientific) followed by size exclusion chromatography. For crystallization, a VSRRLP variant of the elbow region was used to reduce the conformational flexibility between the Fab constant and variable domains [25] .

Purified CR3022 Fab with a VSRRLP modification in the elbow region and SARS-CoV RBD were mixed at a molar ratio of 1:1 and incubated overnight at 4˚C. The complex (7.5 mg/ml) was screened for crystallization using the 384 conditions of the JCSG Core Suite (Qiagen) on our custom-designed robotic CrystalMation system (Rigaku) at Scripps Research by the vapor diffusion method in sitting drops containing 0.1 μl of protein and 0.1 μl of reservoir solution. Optimized crystals were then grown in 2 M sodium chloride and 10% PEG 6000 at 4˚C. Crystals were grown for 7 days and then flash cooled in liquid nitrogen. Diffraction data were collected at cryogenic temperature (100 K) at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2 with a wavelength of 1.033 Å, and processed with HKL2000 [45] . Structures were solved by molecular replacement using PHASER [46] with PDB 6W41 for CR3022 Fab [20] and PDB 2AJF for SARS--CoV RBD [47] . Iterative model building and refinement were carried out in COOT [48] and PHENIX [49] , respectively. Ramachandran statistics were calculated using MolProbity [50] .

Individual mutants for validation experiments were constructed using the QuikChange XL Mutagenesis kit (Stratagene) according to the manufacturer's instructions.

Six molar excess of CR3022 Fab (unmodified) was added to SARS-CoV spike protein 1 hour prior to direct deposition onto carbon-coated 400-mesh copper grids. The grids were stained

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody with 2% (w/v) uranyl-formate for 90 seconds immediately following sample application. Grids were imaged on Tecnai T12 Spirit at 120 keV with a 4k x 4k Eagle CCD. Micrographs were collected using Leginon [51] and images were transferred to Appion [52] for particle picking using a difference-of-Gaussians picker (DoG-picker) [53] and generation of particle stacks. Particle stacks were further transferred to Relion [54] for 2D classification followed by 3D classification to select good classes. Select 3D classes were auto-refined on Relion and used for making figures using UCSF Chimera [55] .

SARS-CoV spike protein was incubated with six molar excess of CR3022 Fab for 2 h. 3.5 μL of the complex (0.9 mg/ml) was mixed with 0.5 μL of 0.04 mM lauryl maltose neopentyl glycol (LMNG) solution immediately before sample deposition onto a 1.2/1.3 300-Gold grid (EMS). The grids were plasma cleaned for 7 seconds using a Gatan Solarus 950 Plasma system prior to sample deposition. Following sample application, grids were blotted for 3 seconds before being vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher).

Data collection was performed using a Talos Arctica TEM at 200 kV with a Gatan K2 Summit detector at a magnification of 36,000x, resulting in a 1.15 Å pixel size. Total exposure was split into 250 ms frames with a total cumulative dose of *50 e -/Å 2 . Micrographs were collected through Leginon software at a nominal defocus range of -0.4 μm to -1.6 μm and MotionCor2 was used for alignment and dose weighting of the frames [51, 56] . Micrographs were transferred to CryoSPARC 2.9 for further processing [57]. CTF estimations were performed using GCTF and micrographs were selected using the Curate Exposures tool in CryoSPARC based on their CTF resolution estimates (cutoff 5 Å) for downstream particle picking, extraction and iterative rounds of 2D classification and selection [58] . Particles selected from 2D classes were transferred to Relion 3.1 for direct C3 refinement, symmetry expansion of particles and iterative rounds of 3D focused classification using spherical masks around the RBD and Fab [54] . Final subsets of clean particles from 4 different classes were each refined with C1 symmetry. Figures were generated using UCSF Chimera and UCSF Chimera X [55] .

Comparisons of subunit rotation angles among different structures were performed with a software 'Superpose' in the CCP4 package [59, 60] . For each classified conformation, the Cα atoms of the RBD domain are superimposed to the equivalent atoms of the RBD in "up"-conformation in a previously reported spike trimer cryo-EM structure (PDB 6ACD) [35] . The rotation matrices generated by superposing each pair of structures with 'Superpose' were adopted to calculate the subunit rotation angle following the equation shown as below:

where θ is the subunit rotation angle, X 11 , Y 22 , and Z 33 represent the X 11 , Y 22 , and Z 33 values in the rotation matrix calculated for the superpose.

Binding assays were performed by biolayer interferometry (BLI) using an Octet Red instrument (FortéBio) as described previously [61] . Briefly, His 6 -tagged RBD proteins at 20 to

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody 100 μg/ml in 1x kinetics buffer (1x PBS, pH 7.4, 0.01% BSA and 0.002% Tween 20) were loaded onto Anti-Penta-HIS (HIS1K) biosensors and incubated with the indicated concentrations of CR3022 Fab. The assay consisted of five steps: 1) baseline: 60 s with 1x kinetics buffer; 2) loading: 300 s with His 6 -tagged S or RBD proteins; 3) baseline: 60 s with 1x kinetics buffer; 4) association: 120 s with samples (Fab or IgG); and 5) dissociation: 120 s with 1x kinetics buffer. For estimating the exact K D , a 1:1 binding model was used.

Pseudovirus preparation and assay were performed as previously described [3] . Briefly, MLVgag/pol and MLV-CMV plasmids was co-transfected into HEK293T cells along with fulllength or P384A SARS-CoV-2 spike plasmids using Lipofectamine 2000 to produce pseudoviruses competent for single-round infection. 

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody Multiple sequence alignment of the RBD sequences was performed by MUSCLE version 3.8.31 [62] . Phylogenetic tree was generated by FastTree version 2.1.8 [63] and displayed by FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). 

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody S8 Fig. Comparison of the angles of approach of CR3022 and COVA1-16 to RBD. The angles of approach of CR3022 (blue) and COVA1-16 (wheat, PDB 7JMW) [39] to RBD are compared. Receptor-binding motif (residues 472-498) on the RBD is colored in pink. (PDF) S1 Table. X-ray data collection and refinement statistics. (PDF) S2 Table. Cryo-EM data collection and refinement statistics. (PDF)

Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

Human neutralizing antibodies elicited by SARS-CoV-2 infection

A neutralizing human antibody binds to the Nterminal domain of the Spike protein of SARS-CoV-2

Broad neutralization of SARS-related viruses by human monoclonal antibodies

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

Potently neutralizing and protective human antibodies against SARS-CoV-2

Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients' B cells

Analysis of a SARS-CoV-2-infected individual reveals development of potent neutralizing antibodies with limited somatic mutation

A human neutralizing antibody targets the receptor binding site of SARS-CoV-2

Potent neutralizing antibodies directed to multiple epitopes on SARS-CoV-2 spike

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

A human monoclonal antibody blocking SARS-CoV-2 infection

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants

Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody

A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV

Cross-neutralization of a SARS-CoV-2 antibody to a functionally conserved site is mediated by avidity

Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies

Bats are natural reservoirs of SARS-like coronaviruses

Locking the elbow: improved antibody Fab fragments as chaperones for structure determination

Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains

Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis

Neutralization of SARS-CoV-2 by destruction of the prefusion spike

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Distinct conformational states of SARS-CoV-2 spike protein

Structures and distributions of SARS-CoV-2 spike proteins on intact virions

Molecular architecture of the SARS-CoV-2 virus

Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

Structural basis of a shared antibody response to SARS-CoV-2

A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction

PLOS PATHOGENS A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody PLOS Pathogens

Cross-neutralization of a SARS-CoV-2 antibody to a functionally conserved site is mediated by avidity

Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody

Recognition of the SARS-CoV-2 receptor binding domain by neutralizing antibodies

Projecting the transmission dynamics of SARS-CoV-2 through the postpandemic period

SARS-CoV-2 vaccines: status report

A highly conserved neutralizing epitope on group 2 influenza A viruses

Processing of X-ray diffraction data collected in oscillation mode

Phaser crystallographic software

Structure of SARS coronavirus spike receptor-binding domain complexed with receptor

Features and development of Coot

PHENIX: a comprehensive Python-based system for macromolecular structure solution

MolProbity: allatom structure validation for macromolecular crystallography

Automated molecular microscopy: the new Leginon system

Appion: an integrated, database-driven pipeline to facilitate EM image processing

DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy

New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife

UCSF Chimera-a visualization system for exploratory research and analysis

MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

Real-time CTF determination and correction

Overview of the CCP4 suite and current developments

Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions

In vitro evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity

MUSCLE: multiple sequence alignment with high accuracy and high throughput

FastTree 2-approximately maximum-likelihood trees for large alignments

We thank Henry Tien for technical support with the crystallization robot, Jeanne Matteson for contribution to mammalian cell culture, Wenli Yu to insect cell culture, Robyn Stanfield for assistance in data collection, and Chris Mok for pilot testing of the pseudovirus assay. We are grateful to the staff of Stanford Synchrotron Radiation Laboratory (SSRL) Beamline 12-2 for assistance.

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

A mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody