key: cord-256572-sqz8yc7b
authors: Huo, Jiandong; Zhao, Yuguang; Ren, Jingshan; Zhou, Daming; Duyvesteyn, Helen ME; Ginn, Helen M; Carrique, Loic; Malinauskas, Tomas; Ruza, Reinis R; Shah, Pranav NM; Tan, Tiong Kit; Rijal, Pramila; Coombes, Naomi; Bewley, Kevin; Radecke, Julika; Paterson, Neil G; Supasa, Piyasa; Mongkolsapaya, Juthathip; Screaton, Gavin R; Carroll, Miles; Townsend, Alain; Fry, Elizabeth E; Owens, Raymond J; Stuart, David I
title: Neutralization of SARS-CoV-2 by destruction of the prefusion Spike
date: 2020-05-06
journal: bioRxiv
DOI: 10.1101/2020.05.05.079202
sha: 
doc_id: 256572
cord_uid: sqz8yc7b

There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. Highlights CR3022 neutralises SARS-CoV-2 Neutralisation is by destroying the prefusion SPIKE conformation This antibody may have therapeutic potential alone or with one blocking receptor attachment

Incursion of animal (usually bat)-derived coronaviruses into the human population has caused several outbreaks of severe disease, starting with severe acute respiratory syndrome (SARS) 3 in 2002 (Menachery et al., 2015) . In late 2019 a highly infectious illness, with cold-like symptoms progressing to pneumonia and acute respiratory failure, resulting in an estimated 6% overall death rate (Baud et al., 2020) , with higher mortality among the elderly and immunocompromised populations, was identified and confirmed as a pandemic by the WHO on 11 th March 2020. The etiological agent is a novel coronavirus (SARS-CoV-2) belonging to lineage B betacoronavirus and sharing 88% sequence identity with bat coronaviruses (Lu et al., 2020a) . The heavily glycosylated trimeric surface Spike protein mediates viral entry into the host cell. It is a large type I transmembrane glycoprotein (the ectodomain alone comprises over 1200 residues) (Wrapp et al., 2020) . It is made as a single polypeptide and then cleaved by host proteases to yield an N-terminal S1 region and the C-terminal S2 region.

Spike exists initially in a pre-fusion state where the domains of S1 cloak the upper portion of the spike with the relatively small (~22 kDa) S1 RBD nestled at the tip. The RBD is predominantly in a 'down' state where the receptor binding site is inaccessible, however it appears that it stochastically flips up with a hinge-like motion transiently presenting the ACE2 receptor binding site (Roy, 2020; Song et al., 2018; Walls et al., 2020; Wrapp et al., 2020) . ACE2 acts as a functional receptor for both SARS-CoV and SARS-CoV-2, binding to the latter with a 10 to 20-fold higher affinity (K D of ~15 nM), possibly contributing to its ease of transmission (Song et al., 2018; Wrapp et al., 2020) . There is 73% sequence identity between the RBDs of SARS-CoV and SARS-CoV-2 ( Figure S1 ). When ACE2 locks on it holds the RBD 'up', destabilising the S1 cloak and possibly favouring conversion to a postfusion form where the S2 subunit, through massive conformational changes, propels its fusion domain upwards to engage with the host membrane, casting off S1 in the process (Song et al., 2018; Wrapp et al., 2020) . Structural studies of the RBD in complex with ACE2 (Lan et al., 2020; Wang et al., 2020b; Yan et al., 2020) how that it is recognized by the extracellular peptidase domain (PD) of ACE2 through mainly polar interactions. The S 4 protein is an attractive candidate for both vaccine development and immunotherapy. Potent nanomolar affinity neutralising human monoclonal antibodies against the SARS-CoV RBD have been identified that attach at the ACE2 receptor binding site (including M396, CR3014 and 80R (Ter Meulen et al., 2006; Sui et al., 2004; Zhu et al., 2007) ). For example 80R binds with nanomolar affinity, prevents binding to ACE2 and the formation of syncytia in vitro, and inhibits viral replication in vivo (Sui et al., 2004) . However, despite the two viruses sharing the same ACE2 receptor these ACE2 blocking antibodies do not bind SARS-CoV-2 RBD (Wrapp et al., 2020) . In contrast CR3022, a SARS-CoV-specific monoclonal selected from a single chain Fv phage display library constructed from lymphocytes of a convalescent SARS patient and reconstructed into IgG1 format (Ter Meulen et al., 2006) , has been reported to cross-react strongly, binding to the RBD of SARS-CoV-2 with a K D of 6.3 nM (Tian et al., 2020) , whilst not competing with the binding of ACE2 (Ter Meulen et al., 2006) . Furthermore, although SARS-CoV escape mutations could be readily generated for ACE2blocking CR3014, no escape mutations could be generated for CR3022, preventing mapping of its epitope (Ter Meulen et al., 2006) . Furthermore a natural mutation of SARS-CoV-2 has now been detected at residue 495 (Y N) (GISAID (Shu and McCauley, 2017) : Accession ID: EPI_ISL_429783 Wienecke-Baldacchino et al., 2020), which forms part of the ACE2 binding epitope. Finally, CR3022 and CR3014 act synergistically to neutralise SARS-CoV with extreme potency (Ter Meulen et al., 2006) . Whilst this work was being prepared for publication a paper reporting that CR3022 does not neutralise SARS-CoV-2 and describing the structure of the complex with the RBD at 3.1 Å resolution was published (Yuan et al., 2020) . Here we extend the structure analysis to significantly higher resolution and, using a different neutralisation assay, show that CR3022 does neutralise SARS-CoV-2, but via a mechanism that would not be detected by the method of Yuan et al (Yuan et al., 2020) . We use cryo-EM analysis of the interaction of CR3022 with the full Spike ectodomain to confirm 5 this mechanism. Taken together these observations suggest that the CR3022 epitope should be a major target for therapeutic antibodies.

To understand how CR3022 works we first investigated the interaction of CR3022 Fab with isolated recombinant SARS-CoV-2 RBD, both alone and in the presence of ACE2. Surface plasmon resonance (SPR) measurements (Methods and Figure S2 ) confirmed that CR3022 binding to RBD is strong (although weaker than the binding reported to SARS-CoV (Ter Meulen et al., 2006) ), with a slight variation according to whether CR3022 or RBD is used as the analyte (K D = 30 nM and 15 nM respectively, derived from the kinetic data in Table S1 ).

An independent measure using Bio-Layer Interferometry (BLI) with RBD as analyte gave a K D of 19 nM (Methods and Figure S2 ). These values are quite similar to those reported by Tian et al. (Tian et al., 2020 ) (6.6 nM), whereas weaker binding (K D ~ 115 nM) was reported recently by Yuan et al. (Yuan et al., 2020) . Using SPR to perform a competition assay revealed that the binding of ACE2 to the RBD is perturbed by the presence of CR3022 ( Figure S3 ). The presence of ACE2 slows the binding of CR3022 to RBD and accelerates the dissociation. Similarly, the release of ACE2 from RBD is accelerated by the presence of CR3022. These observations are suggestive of an allosteric effect between ACE2 and CR3022.

A plaque reduction neutralisation test using SARS-CoV-2 virus and CR3022 showed an ND50 of 1:201 for a starting concentration of 2mg/mL (calculated according to Grist (Grist, 1966) ), superior to that of MERS convalescent serum (ND50 of 1:149) used as a NIBSC 6 international standard positive control (see Methods and Table S2 ). This corresponds to 50% neutralisation at ~70 nM (~10.5 ug/mL). This is similar to the neutralising concentration (50% neutralisation at 11 ug/mL) reported by Ter Meulen et al. (Ter Meulen et al., 2006) for SARS-CoV, however, as discussed below, it is in apparent disagreement with the result reported recently by Yuan et al. (Yuan et al., 2020) .

We determined the crystal structure of the SARS-CoV-2 RBD-CR3022 Fab complex (see Methods and Table S3 ) to investigate the relationship between the binding epitopes of ACE2 and CR3022. Crystals grew rapidly and consistently. Two crystal forms grew in the same drop. The solvent content of the crystal form solved first was unusually high (ca 87%) with the ACE2 binding site exposed to large continuous solvent channels within the crystal lattice ( Figure S4 ). These crystals therefore offer a promising vehicle for crystallographic screening to identify potential therapeutics that could act to block virus attachment. The current analysis of this crystal form is at 4.4 Å resolution and so, to avoid overfitting, refinement used a novel real-space refinement algorithm to optimise the phases (Vagabond, HMG unpublished, see Methods). This, together with the favourable observation to parameter ratio resulting from the exceptionally high solvent content, meant that the map was of very high quality, allowing reliable structural interpretation ( Figure S5 , Methods). Full interpretation of the detailed interactions between CR3022 and the RBD was enabled by the second crystal form which diffracted to high resolution, 2.4 Å, and the structure of which was refined to give an R-work/R-free of 0.213/0.239 and good stereochemistry (Methods, Table S3, Figure   S5 ).

The high-resolution structure is shown in Figure 1a . There are two complexes in the crystal asymmetric unit with residues 331-529 in one RBD, 332-445 and 448-532 in the other RBD well defined, whilst residues133-136 of the CR3022 heavy chains are disordered. The RBD has a very similar structure to that seen in the complex of SARS-CoV-2 RBD with ACE2, rmsd for 194 Ca atoms of 0.6 Å 2 (PDB, 6M0J (Lan et al., 2020) ), and an rmsd of 1.1 Å 2 compared to the SARS CoV RBD (PDB, 2AJF (Li et al., 2005) ). Only minor conformational changes are introduced by binding to CR3022, at residues 381-390. The RBD was deglycosylated (Methods) to leave a single saccharide unit at each of the N-linked glycosylation sites clearly seen at N331 and N343 ( Figure S5 ). CR3022 attaches to the RBD surface orthogonal to the ACE2 receptor binding site. There is no overlap between the epitopes and indeed both the Fab and ACE2 ectodomain can bind without clashing ( Figure   1d ) (Tian et al., 2020) . Such independence of the ACE2 binding site has been reported recently for another SARS-CoV-2 neutralising antibody, 47D11 . The Fab complex interface buries 990 Å 2 of surface area (600 and 390 Å 2 by the heavy and light chains respectively, Figure 2a and Figure S6 ), somewhat more than the RBD-ACE2 interface which covers 850 Å 2 (PDB 6M0J (Lan et al., 2020) ). Typical of a Fab complex, the interaction is mediated by the antibody CDR loops, which fit well into the rather sculpted surface of the RBD (Figure 1b , c). The heavy chain CDR1, 2 and 3 make contacts to residues from α 2, β 2 and α 3 (residues 369-386), while two of the light chain CDRs (1 and 2) interact mainly with residues from the β 2-α3 loop, α 3 (380-392) and the α 5-β4 loop (427-430) (Figures 1, S1, S7). A total of 16 residues from the heavy chain and 14 from the light chain cement the interaction with 26 residues from the RBD. For the heavy chain these potentially form 7 H-bonds and 3 salt bridges, the latter from D55 and E57 (CDR2) to K378 of the RBD.

Whilst the light chain interface comprises 6 H-bonds and a single salt bridge between E61

(CDR2) and K386 of the RBD. The binding is consolidated by a number of hydrophobic 8 interactions ( Figure S7b ). Of the 26 residues involved in the interaction 23 are conserved between SARS-CoV and SARS-CoV-2 ( Figure 2b and Figure S1 ). The CR30222 epitope is much more conserved than that of the receptor blocking anti-SARS-CoV antibody 80R for which only 13 of the 29 interacting residues are conserved (Hwang et al., 2006) , in-line with the lack of cross reactivity observed for the latter.

The reason for the conservation of the CR3022 epitope becomes clear in the context of the complete pre-fusion S structure (PDB IDs: 6VSB (Wrapp et al., 2020) , 6VXX, 6VYB (Walls et al., 2020) ) where the epitope is inaccessible ( Figure 3 ). When the RBD is in the 'down'

configuration the CR3022 epitope is packed tightly against another RBD of the trimer and the N-terminal domain (NTD) of the neighbouring protomer. In the structure of the pre-fusion form of trimeric Spike the majority of RBDs are 'down', although presumably stochastically one may be 'up' (Walls et al., 2020; Wrapp et al., 2020) . The structure of a SARS-CoV complex with ACE2 ectodomain shows that this 'up' configuration is competent to bind receptor, and that there are a family of 'up' orientations with significantly different hinge angles (Song et al., 2018) . However, the CR3022 epitope remains largely inaccessible even in the 'up' configuration. Modelling the rotation of the RBD required to enable Fab interaction in the context of the Spike trimer, showed a rotation corresponding to a > 60° further declination from the central vertical axis was required, beyond that observed previously (Walls et al., 2020; Wrapp et al., 2020) (Figure 3i ), although this might be partly mitigated by more complex movements of the RBD and if more than one RBD is in the 'up' configuration this requirement would be relaxed somewhat. Since locking the up state by receptor blocking antibodies is thought to destabilise the pre-fusion state (Walls et al., 2019) binding of CR3022 presumably introduces further destabilisation, leading to a premature conversion to the post-fusion state, inactivating the virus. CR3022 and ACE2 blocking antibodies can bind 9 independently but both induce an 'up' conformation, presumably explaining the observed synergy between binding at the two sites (Ter Meulen et al., 2006) .

To test if CR3022 binding destabilises the prefusion state of Spike, the ectodomain construct described previously (Wrapp et al., 2020) was used to produce glycosylated protein in HEK cells (Methods). Cryo-EM screening showed that the protein was in the trimeric prefusion conformation. Spike was then mixed with an excess of CR3022 Fab and incubated at room temperature, with aliquots being taken at 50 minutes and 3 hours. Aliquots were immediately applied to cryo-EM grids and frozen (Methods). For the 50 minutes incubation, collection of a substantial amount of data allowed unbiased particle picking and 2D classification which revealed two major structural classes with a similar number in each, (i) the prefusion conformation, and (ii) a radically different conformation (Methods , Table S4 and Figure S8 ).

Detailed analysis of the prefusion conformation led to a structure at a nominal resolution of 3.4 Å (FSC = 0.143), based on a broad distribution of orientations, that revealed the same predominant RBD pattern (one 'up' and two 'down') previously seen (Wrapp et al., 2020) with no evidence of CR3022 binding (Figure 4a , Figure S9 ). Analysis of the other major particle class revealed strong preferential orientation of the particles on the grid ( Figure   S10a ). Despite this a reconstruction with a nominal resolution of 3.9 Å within the plane of the grid, and perhaps 7 Å resolution in the perpendicular direction ( Figure S10b ), could be produced which allowed the unambiguous fitting of the CR3022-RBD complex (Figure 4b ).

Note that in addition there is less well defined density attached to the RBD, in a suitable position to correspond to the Spike N-terminal domain (Wrapp et al., 2020) . These structures are no longer trimeric, rather two complexes associate to form an approximately symmetric dimer (however, application of this symmetry in the reconstruction process did not improve 1 0 the resolution). The interactions responsible for dimerisation involve the ACE2 binding site on the RBD and the elbow of the Fab, however the interaction does not occur in our lowresolution crystal form and is therefore probably extremely weak and not biologically significant. Since conversion to the post-fusion conformation leads to dissociation of S1

(which includes the N-terminal domain and RBD) these results confirm that CR3022 destabilises the prefusion Spike conformation. Further evidence of this is provided by analysis of data collected after 3 h incubation. By this point there were no intact trimers remaining and a heterogeneous range of oligomeric assemblies had appeared, which we were not able to interpret in detail but which are consistent with the lateral assembly of Fab/RBD complexes ( Figure S11 ). Note that the relatively slow kinetics will not be representative of events in vivo, where the conversion might be accelerated by the elevated temperature and the absence of the mutations which were added to this construct to stabilise the prefusion state (Kirchdoerfer et al., 2018; Pallesen et al., 2017; Wrapp et al., 2020) .

Until now the only documented mechanism of neutralisation of coronaviruses has been through blocking receptor attachment. In the case of SARS-CoV this is achieved by presentation of the RBD of the Spike in an 'up' conformation.

Although not yet confirmed for SARS-CoV-2 it is very likely that a similar mechanism can apply. Here we define a second class of neutralisers, that bind a highly conserved epitope ( Figure S1 ) and can therefore act against both SARS-CoV and SARS-CoV-2 (CR3022 was first identified as a neutralising antibody against SARS-CoV (Ter Meulen et al., 2006) ). We find that binding of CR3022 to the isolated RBD is tight (~20 nM) and the crystal structure of the complex reveals the atomic detail of the interaction. Despite the spatial separation of the CR3022 and ACE2 epitopes we find an allosteric effect between the two binding events. The role of the 1 1 CR3022 epitope in stabilising the prefusion Spike trimer explains why it has, to date, proved impossible to generate mutations that escape binding of the antibody (Ter Meulen et al., 2006) .

Whilst in our assay CR3022 neutralises SARS-CoV-2, a recent paper (Yuan et al., 2020) reported an alternative assay that did not detect neutralisation. The difference is likely due to their removal of the antibody/virus mix after adsorption to the indicator cells, before incubating to allow cytopathic effect (CPE) to develop. This would be in-line with the distinction previously seen between neutralisation tests for influenza virus by antibodies which bind the stem of hemagglutinin and therefore do not block receptor binding (Thomson et al., 2012) . These antibodies did not appear to be neutralising when tested with the standard WHO neutralisation assay, in which a similar protocol is used to that adopted by Yuan et al, in which the inoculum of virus/antibody is washed out before development of CPE.

Neutralisation was observed, however, when the antibodies were left in the assay during incubation to produce CPE. By analogy we would expect antibodies to the RBD that block attachment to ACE2 to behave in a similar way to antibodies against the globular head of HA, whilst antibodies such as CR3022, that neutralise by an alternative mechanism to blocking receptor attachment, may need to be present throughout the incubation period with the indicator cells to reveal neutralisation. This agrees with our observation that, in the absence of ACE2, the CR3022 Fab destroys the prefusion-stabilised trimer (T 1/2 ~1h at room temperature as measured by cryo-EM).

With monoclonal antibodies now recognised as potential antivirals (Lu et al., 2020b; Salazar et al., 2017) our results suggest that CR3022 may be of immediate utility, since the mechanism of neutralisation will be unusually resistant to virus escape. In contrast antibodies which compete with ACE2 (whose epitope on SARS-CoV-2 is reported to have already 1 2

shown mutation at residue 495 (GISAID: Accession ID: EPI_ISL_429783 Wienecke-Baldacchino et al., 2020 (Shu and McCauley, 2017) ), are likely to be susceptible to escape.

Furthermore, with knowledge of the detailed structure of the epitope presented here a higher affinity version of CR3022 might be engineered. Alternatively, since the same mechanism of neutralisation is likely to be used by other antibodies, a more potent monoclonal antibody targeting the same epitope might be found (for instance by screening for competition with CR3022). Additionally, since this epitope is sterically and functionally independent of the well-established receptor-blocking neutralising antibody epitope there is considerable scope for therapeutic synergy between antibodies targeting the two epitopes (indeed this type of 

To further validate the SPR results the K D of Fab CR3022 for RBD was also measured by bio-layer interferometry. Kinetic assays were performed on an Octet Red 96e (ForteBio) at 30 ℃ with a shake speed of 1000 rpm. Fab CR3022 was immobilized onto amine reactive 2nd generation (AR2G) biosensors (ForteBio) and serially diluted RBD (80，40，20，10 and 5 nM) was used as analyte. PBS (pH 7.4) was used as the assay buffer. Recorded data were analysed using the Data Analysis Software HT v11.1 (Fortebio), with a global 1:1 fitting model.

Neutralising virus titres were measured in serum samples that had been heat-inactivated at 56 °C for 30 minutes. SARS-CoV-2 (strain Victoria/1/2020 at cell passage 3 (Caly et al., 2020) ) was diluted to a concentration of 1.4E+03 pfu/mL (70 pfu/50 µl) and mixed 50:50 in 1% FCS/MEM containing 25 mM HEPES buffer with doubling serum dilutions from 1:10 to 1:320 in a 96-well V-bottomed plate.

The plate was incubated at 37 °C in a humidified box for 1 hour to allow the antibody in the serum samples to neutralise the virus. CR3022 (pH7.2) at a starting concentration of 2 mg/mL was diluted 1 in 10. The dilutions were then made 2-fold up to 320. The neutralised virus was transferred into the wells of a twice DPBS-washed plaque assay 24-well plate that had been seeded with Vero/hSLAM the previous day at 1.5E+05 cells per well in 10% FCS/MEM. Neutralised virus was allowed to adsorb at 37 °C for a further hour, and overlaid with plaque assay overlay media (1X MEM/1.5% CMC/4% FCS final). After 5 days incubation at 37 °C in a humified box, the plates were fixed, stained and plaques counted.

Dilutions and controls were performed in duplicate. Median neutralising titres (ND50) were determined using the Spearman-Karber formula (Kärber, 1931) relative to virus only control wells.

Purified and deglycosylated RBD and CR3022 Fab were concentrated to 8.3 mg/mL and 11 mg/mL respectively, and then mixed in an approximate molar ratio of 1:1. Crystallization screen experiments were carried out using the nanolitre sitting-drop vapour diffusion method in 96-well plates as previously described (Walter et al., 2003 (Walter et al., , 2005 transmission). Data were indexed, integrated and scaled with the automated data processing program Xia2-dials (Winter, 2010; Winter et al., 2018) . The data set of 720° was collected from a single frozen crystal to 4.4 Å resolution with 52-fold redundancy. The crystal belongs to space group P4 1 2 1 2 with unit cell dimensions a = b = 150.5 Å and c = 241.6 Å. The structure was determined by molecular replacement with PHASER (McCoy et al., 2007) using search models of human germline antibody Fabs 5-51/O12 (PDB ID, 4KMT (Teplyakov et al., 2014) ) heavy chain and IGHV3-23/IGK4-1 (PDB ID, 5I1D (Teplyakov et al., 2016) ) light chain, and RBD of SARS-CoV-2 RBD/ACE2 complex (PDB ID, 6M0J (Lan et al., 2020) ). There is one RBD/CR3022 complex in the crystal asymmetric unit, resulting in a crystal solvent content of ~87%.

During optimization of the crystallization conditions, a second crystal form was found to grow in the same condition with similar morphology. A data set of 720° rotation with data extending to 2.4 Å was collected on beamline I03 of Diamond from one of these crystals (exposure time 0.004 s per 0.1° frame, beam size 80×20 μ m and 100% beam transmission).

The crystal also belongs to space group P4 1 2 1 2 but with significantly different unit cell dimensions (a = b = 163.1 Å and c = 189.1 Å). There were two RBD/CR3022 complexes in the asymmetric unit and a solvent content of ~74%.

The initial structure was determined using the lower resolution data from the first crystal form. Data were excluded at a resolution below 35 Å as these fell under the beamstop shadow. One cycle of REFMAC5 (Murshudov et al., 2011) was used to refine atomic coordinates after manual correction in COOT (Emsley and Cowtan, 2004) Figure S5 ). The final refined structure had an R work of 0.331 (R free , 0.315) for all data to 4.36 Å resolution. This structure was later used to determine the structure of the second crystal form, which has been refined with PHENIX (Liebschner et al., 2019) to R work = 0.213 and R free = 0.239 for all data to 2.42 Å resolution. This refined model

revealed the presence of one extra residue at each heavy chain N-terminus and 3 extra residues at the N-terminus of one RBD from the signal peptide. There is well ordered density for a single glycan at each of the glycosylation sites at N331 and N343 in one RBD, and only one at N343 in the second RBD.

Data collection and structure refinement statistics are given in Table S3 . Structural comparisons used SHP (Stuart et al., 1979) , residues forming the RBD/Fab interface were identified with PISA (Krissinel and Henrick, 2007) , figures were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).

Purified spike protein was buffer exchanged into 2 mM Tris pH 8.0, 200 mM NaCl, 0.02 % NaN3 buffer using a desalting column (Zeba, Thermo Fisher μ m and at a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel, see Table S4 .

Cryo-EM data processing 2 2

For both the 50 minute and 3 h incubation datasets, motion correction and alignment of 2x binned super-resolution movies was performed using Relion3.1. CTF-estimation with GCTF (v1.06) (Zhang, 2016) and non-template-driven particle picking was then performed within cryoSPARC v2.14.1-live followed by multiple rounds of 2D classification (Punjani et al., 2017) .

For the 50 minutes dataset. 2D class averages for structure-A and structure-B were then used separately for template-driven classification before further rounds of 2D and 3D classification with C1 symmetry. Both structures were then sharpened in cryoSPARC. Data processing and refinement statistics are given in Table S4 .

An initial model for the spike (structure-A) was generated using PDB ID, 6VYB (Walls et al., 2020) and rigid body fitted into the final map using COOT (Emsley and Cowtan, 2004) .

The model was further refined in real space with PHENIX (Liebschner et al., 2019) which resulted in a correlation coefficient of 0.84. Two copies of RBD-CR3022 were fitted into structure-B in the same manner. Because of the strongly anisotropic resolution the overall correlation coefficient vs the model was lower (0.47).

For the 3 h incubation dataset, particles were extracted with a larger box size (686 pixels as compared to 540 pixels), and, following multiple rounds of 2D classification, 2D class averages from 'blob-picked' particles showing signs of complete 'flower-like' structures were selected for ab initio reconstruction. For the 3 h data no detailed fitting was attempted. T/e (red, negative; blue, positive).

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RBDs in the down conformation (generated by superposing our RBD structure on the prefusion trimer of ref (Wrapp et al., 2020) ). The viral membrane would be at the bottom of the picture. All of S1 and S2 are shown in yellow, apart from the RBD, which is shown in grey, with the CR3022 epitope coloured green. a, A cut-way of the trimer showing, in red, the dipeptide (residues 986-987) which has been mutated to PP to confer stability on the pre-fusion state. Note the proximity to the CR3022 epitope. c, Showing a top view of the molecule (also used for panels d-f). One of the RBDs has been drawn in light grey in the down configuration 2 8 and hinged up in dark grey, using the motion about the hinge axis observed for several coronavirus Spikes, but extending the motion sufficiently to allow CR3022 to bind. The PP motif is shown in red and the glycosylated residue N343 in magenta. Panels d-f show the trimer viewed from above d -all RBDs down, e -one RBD up f -one RBD rotated (as in c)to allow access to CR3022. Panels g-i are equivalent structures to d-f, but are viewed from the side. In e bound ACE2 is shown and in f CR3022.