key: cord-0293878-pm10w02w
authors: Castelli, Matteo; Scietti, Luigi; Clementi, Nicola; Cavallaro, Mattia; Faravelli, Silvia; Pinnola, Alberta; Criscuolo, Elena; Diotti, Roberta Antonia; Clementi, Massimo; Forneris, Federico; Mancini, Nicasio
title: SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host
date: 2021-08-11
journal: bioRxiv
DOI: 10.1101/2021.08.11.455960
sha: 4e4090bb35b931ea386f2e840360846b6b0aaa5a
doc_id: 293878
cord_uid: pm10w02w

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 – the closest bat ancestor – spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.

The Coronaviridae family comprises seven species of human interest; four are endemic and highly adapted to humans (HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1), two epidemic (MERS-CoV and SARS-CoV-1) and one pandemic (SARS-CoV-2). Except for HCoV-OC43 and HCoV-HKU1, their ancestral origin can be traced back to coronaviruses (CoV) infecting bats, the main natural host reservoir of α-and β-coronavirus genera (1-3).

SARS-CoV-1 and MERS-CoV are β-CoVs characterized by low inter-human transmission and high fatality rate, indicative of a zoonotic infection and a sub-optimal adaptation to humans (4) . Both viruses gained the ability to infect humans following adaptation in a putative intermediate host, although with different evolutionary trajectories. While MERS-CoV ancestors stably adapted to dromedary camels decades ago diverging from bat MERSrelated viruses, SARS-CoV-1 direct ancestor seems to have transiently jumped from horseshoe bats (Rhinolophus spp.) to palm civets and/or raccoon dogs, accumulating a few mutations that incidentally increased its ability to infect humans.

SARS-CoV-2 was first identified in the city of Wuhan in December 2019 and rapidly spread worldwide due to a high inter-human transmission rate and a relevant percentage of asymptomatic and paucisymptomatic infections (5, 6) . Phylogenetic analysis identified SARS-CoV-2 as a member of a novel clade in the Sarbecovirus lineage that also comprises viruses retrospectively identified in Southeast Asian pangolins (Manis javanica and Manis pentadactila) and horseshoe bats (7) (8) (9) . Among them, RaTG13, collected in 2013 from a R.

affinis specimen in China's Yunnan province, shows the highest homology to SARS-CoV-2 both genome-wide and at the spike gene level (5, 10) . As such, RaTG13 is to date the closest relative of SARS-CoV-2, thus supporting its bat origin. In analogy with SARS-CoV-1 and several bat SARS-related (SARSr) viruses, both RaTG13 and SARS-CoV-2 engage the host angiotensin-converting enzyme 2 (ACE2) to mediate cell entry despite the high sequence divergence at the receptor binding domain (RBD) (11, 12) .

A virus spillover probability is hardly predictable but, in principle, it is directly proportional to the phylogenetic distance between donor and recipient species. In this context, adaptation in an intermediate host may serve to lower the species barrier. Compared to other viruses, CoVs can jump among host species with relative ease and the major tropism determinant is represented by the spike ability to mediate entry, in turns mainly dependent on the host receptor orthologues conservation. Under this perspective, ACE2 differences between humans and R. affinis argue against a direct spillover event. Indeed, albeit still competent, RaTG13 spike binds to human ACE2 (hACE2) and mediates pseudotyped virus entry at a lower extent than SARS-CoV-2 (12, 13) . While this favors the hypothesis of an intermediate host, no evidence of it have emerged so far, leaving several unanswered questions on the evolutionary path followed by SARS-CoV-2 and posing major concerns on the possible emergence of related viruses with pandemic potential (14) .

To trace SARS-CoV-2 evolutionary trajectory, we used a combination of surface plasmon resonance (SPR), X-ray crystallography and molecular dynamics (MD) simulation-based techniques to characterize the functional features of RaTG13 and SARS-CoV-2 spikes.

Despite sequence divergence, we found that both RBDs engage hACE2 with nearly identical binding mode and affinity. Furthermore, we measured comparable affinities in the nanomolar range also for R. affinis ACE2 (affiACE2). At the spike level, SARS-CoV-2 is significantly more optimized to expose the RBD in the conformation competent to ACE2 binding and mutations in all domains contribute to it. Taken together, our results point out to an evolutionary process that regarded exclusively the spike dynamics and kinetics through the fine-tuning of the pre-fusion states metastability. Also, RaTG13 and SARS-CoV-2 RBD promiscuity rules out the requirement of an intermediate host to lower the species barrier.

R. affinis ACE2 known coding sequences (GenBank database accession IDs MT394203 to MT394225) were fed to Sequence Read Archive (SRA) Nucleotide BLAST to retrieve ACE2 Illumina reads from the SRA dataset with accession ID SRX7724752. Reads were assembled in contigs with Spades (34) .

The pCAGGS plasmids for production of the C-terminal His-tagged SARS-CoV-2 S protein (#NR_52310) and RBD (#NR_52310), were obtained from BEIRESOURCES (NY, USA).

The designed codon-optimized sequence encoding for RaTG13 RBD and the cDNA for The Netherlands) providing the human cystatin protein signal peptide and C-terminal 6xHistag for purification. The cDNA for hACE2 ectodomain was obtained from AddGene (#141185). The sequence was amplified using PCR with oligonucleotides hACE2ecto-Fw 

Recombinant hACE2 ectodomain, affiACE2 ectodomain, SARS-CoV-2 RBD, and RaTG13

RBD were produced using HEK293-F cells (Invitrogen) cultivated in suspension using

Freestyle medium (Invitrogen) as described in (35 RaTG13 RBD with concentration ranging from 250 to 7.8 nM were prepared in the running buffer (25 mM HEPES/NaOH, 150 mM NaCl and 0.05% Tween-20, pH 7.2) and injected using a flow of 50 μl min −1 . Analysis was performed using the Biacore evaluation software (GE Healthcare) using a 1:1 affinity model.

RaTG13 RBD and hACE2 were mixed in a molar ratio of 1:1. 3 MicroMounts Loops (Mitegen), cryo-protected with the mother liquor supplemented with 20% glycerol, flash-cooled and stored in liquid nitrogen prior to data acquisition.

Diffraction data from hACE2-RaTG13 RBD crystals were collected at the ID23-1 beamline of the European Synchrotron Radiation Facility (ESRF, Grenoble, FR). Data were integrated using the automatic XIA2-DIALS pipeline available at the beamline outstation and scaled with AIMLESS (36, 37) . Data collection statistics are summarized in Table S1 . Individual search models for hACE2 and SARS-CoV-2 RBD molecules were extracted from PDB 6VW1 using COOT and used for molecular replament with PHASER, yielding complete solutions for both crystal forms comprising two hACE2 and two RBD molecules in asymmetric unit (38, 39) .

Final 3D models were generated using iterations of automatic refinement using low resolution protocols in phenix.refine using non crystallographic symmetry restraints as well as external restraints based on individual chains from PDB ID 6VW1, alternated with manual adjustments using COOT (40) . Assessment of final structure quality was carried out with the Molprobity server and with the RCSB PDB Validation Server (39, 41) . Final refinement statistics are listed in Table S1 .

Deglycosylated spike ectodomains (residues 14-1146) were completed, reverted to the wildtype when needed or mutated with Modeller using the following templates: PDB ID 6ZGF

for RaTG13 closed, PDB ID 6ZGG for all cleaved, 1-up systems, PDB ID 6ZGI for all cleaved, closed systems, PDB ID 6VYB for all uncleaved, 1-up system and PDB ID 6ZGE for all uncleaved, closed systems (42) . To reconstruct the structure of RaTG13 spike complexed with hACE2, the final frames of the RaTG13 1-up state and RaTG13

RBD/hACE2 simulations were used as templates in Modeller. All structures were simulated in an orthorhombic TIP3P water box, neutralized with the proper counterions, and parametrized using the all-atom AMBER/parm12SB force field (43) . All simulations were performed using the GROMACS 5.1.4 code (44) . Periodic boundary conditions in the three axes were applied. Covalent bond length, including hydrogen bonds, was set using the LINCS algorithm, allowing a time-integration step of 2 fs. Constant pressure was imposed using the Parrinello-Rahman barostat with a time constant of 2 ps and a reference pressure of 1 bar, while the constant temperature was maintained using the modified Berendsen thermostat with a time constant of 0.1 ps. Long-range electrostatic interactions were calculated with the particle mesh Ewald method with a real-space cutoff of 12 Å. Each system was minimized with the steepest descent algorithm, equilibrated for 100 ps in an NVT ensemble followed by 100 ps in an NPT ensemble, and then subject to a 200 ns simulation at constant temperature (300 K).

RBDs contacts were characterized by calculating inter-protomer dynamic cross-correlation and contact map (45) . RBDs geometry along the trajectories was measured as RBD rotation 

Several bat SARSr viruses have the ability to recognize hACE2, surprisingly often at higher affinity than bat ACE2, without prior adaptation (15) . The marked sequence differences between RaTG13/SARS-CoV-2 RBDs (89.2% amino acid identity) and human/R.

affinis ACE2 (80.7% identity), and in particular those found at the RBD-ACE2 interface ( Fig.   1A ), suggest high species barriers to efficient binding and therefore the need of adaptation in an intermediate host.

To verify this hypothesis, we first measured the affinity of RaTG13 and SARS-CoV-2 RBDs for hACE2 by SPR. Surprisingly, we found that both RBDs bind to hACE2 with K d in the nanomolar range (21 and 41 nM, respectively) ( Fig. 2A) . Prompted by this observation, we attempted crystallization of the RaTG13 RBD/hACE2 complex, obtaining crystals suitable for diffraction experiments in two distinct crystal forms (Table   S1 ). After structure determination using molecular replacement, crystal form 1 yielded a 4.5

Å resolution structure of the RaTG13/RBD-hACE2 complex (Fig. 2B) showing identical crystal packing assembly to that of the previously determined SARS-CoV-2 RBD/hACE2 complex, whereas crystal form 2 could be solved at 6.5-Å resolution, revealing a different crystal packing assembly (Fig. S1 ) (12) . Both RaTG13 RBD/hACE2 structures are superimposable to SARS-CoV-2 RBD/hACE2, with minor adjustments associated to the amino acid differences. Therefore, sequence differences between RaTG13 and SARS-CoV-2 RBD do not affect the binding mode nor the affinity for hACE2.

To further characterize the evolutionary trajectory of SARS-CoV-2, we next measured RBDs affinity for affiACE2. Several affiACE2 sequences were recently deposited and show moderate variability, with eight polymorphic positions (15) . We mined the original raw sequencing dataset RaTG13 was identified from and uniquely determined that the specific R.

affinis specimen carried a minority allele, characterized by the H34, D38 -both lying at the RBD/ACE2 interface -and A185 polymorphisms (Fig. 1B) . SPR measurements show

RaTG13 and SARS-CoV-2 RBD affinities for affiACE2 almost identical to hACE2 (12 and 18 nM, respectively) ( Fig. 2A) . Thus, in terms of RBD affinity, the species barrier between R.

affinis bats and humans is negligible for both RaTG13 and SARS-CoV-2, strongly supporting the possibility of a direct species jump from bats to humans of SARS-CoV-2 and related viruses. As a consequence, SARS-CoV-2 might have directly evolved in R. affinis bats.

Our results indicate that the affinity of RaTG13 and SARS-CoV-2 RBD for hACE2 is equivalent. However, when the entire spike is considered, SARS-CoV-2 is a significantly better hACE2 binder and mediates pseudotyped virus entry more efficiently (12, 13) .

RaTG13 spike cryoEM structures show exclusively the closed state, while SARS-CoV-2 uncleaved, S0 form is found also in the 1-up state (one RBD exposed and two closed),

suggesting that different functional properties might be related to the spike propensity of exposing the RBD for ACE2 engagement. To verify this hypothesis, we performed full-atom, RBD required to accommodate the closure movement -followed by a rapid RBD closure (Fig. 4) . Conversely, the closed-to-1up transitions follow markedly different paths: as a consequence of the closed RBDs tight interactions, RaTG13 opening is slow and geometrically almost linear, while SARS-CoV-2 S0 is faster and follows a biphasic transition. Its kinetics presents a preparatory initial phase where the RBD motion changes slowly and a subsequent abrupt switch to the open state, indicating that the spike reached a conformation where the RBD is free to move. Altogether, the mutations from RaTG13 to SARS-CoV-2 affect the entire spike pre-fusion states metastability and, by optimizing the RBDs angle of exposure and transitions kinetics, they increase infectivity regardless of the direct affinity for ACE2.

Besides the 21 mutations in the RBD, RaTG13 and SARS-CoV-2 spikes differ on four positions in the NTD, one in each subdomain 1 and 2 (SD1 and SD2, respectively), two in S2

(of which only S1125N is comprised in the cryoEM structures and our systems) and the furin cleavage site, a four-residue insertion ( 681 PRRA 684 ) at the S1/S2 junction (Fig. 5A) . Hence, the dramatic differences between RaTG13 and SARS-CoV-2 spike dynamics may be due to the demonstrating its residual functionality but also a markedly lower fitness (17) (18) (19) . The 1-up state of RaTG13 RBD and SARS-CoV-2 ΔPRRA shows large RBD opening (Ψ ~130° and ~120°, respectively) and rotation angles (Φ ~50° and ~60°, respectively) (Fig. 5B) . Similar Ψ values are found in SARS-CoV-2 cryoEM structures, but Φ values are always below ~35°, suggesting a functional limit to RBD rotation (20, 21) . To confirm this hypothesis, we applied a steered molecular dynamics (SMD) protocol to SARS-CoV-2 3-up spike fully engaged with ACE2 to drive all RBDs to Φ = 60°. Reaching the target rotation angle indeed also causes large Ψ increases (up to 165°) and dramatic S1 rearrangements (Fig. S7) , thus relating SARS-CoV-2 ΔPRRA low fitness to prevented furin cleavage and disrupted spike conformation. Conversely, RaTG13 RBD+PRRA has an opening angle comparable to SARS-CoV-2 S0 and a remarkably low rotation (Φ ~-10°). Since we recently characterized a SARS-CoV-2 clinical isolate with analogous Φ angle and driving the 3-up state to this Φ value in SMD does not alter the spike global conformation (Fig. S7) , we can speculate that RaTG13 RBD+PRRA geometry is functional (22) . Therefore, we next analyzed its closed-to-1up transition in TMD and identified a biphasic kinetics similar to SARS-CoV-2 S0, although smoother and slower (Fig. S8) . The hybrid systems involving SARS-CoV-2 RBD confirm that the sequence of this domain dominates the spike dynamics and kinetics. However, the inclusion of mutations in other domains also highlights relevant allosteric effects. In support to this, structural inspection of RaTG13 RBD and SARS-CoV-2 ΔPRRA trajectories shows that the RBD large aperture is stabilized by the N519H RBD mutation, as the histidine stably inserts into a hydrophobic pocket created by residues V130, F168, L229, I231 and I233 (Fig. S9) 

The furin cleavage site at the S1/S2 junction is found in several unrelated bat βcoronaviruses and a few SARS-CoV-2-related viruses bearing partial furin cleavage site have been recently identified in horseshoe bats, supporting the possibility it arose along the evolution in R. affinis (7, 8, 23) . While the most likely selective advantage of incorporating it comes from a spike already primed for fusion during biogenesis, cryoEM structures of SARS-CoV-2 S1/S2, cleaved spike demonstrated also a higher propensity to expose the RBDs than the S0 form. To better explore the molecular details associated to furin cleavage and hypothesize when and how the cleavage site was incorporated, we ran unbiased MD simulations of the cleaved, S1/S2 spike form of all wild-type and hybrid systems bearing the 681 PRRA 684 sequence (Fig. 5) .

Compared to the uncleaved form, SARS-CoV-2 S1/S2 1-up state open RBD is in a more upright position achieved through a balanced increase in opening and rotation angles. This conformation is highly similar to that of the ACE2-bound, cleaved spike structure described by Benton et al., demonstrating that ACE2 acts by stabilizing rather than inducing it (20) .

The transitions simulated in TMD show slower 1-up-to-closed and faster closed-to-1up kinetics compared to the uncleaved form. The latter transition is improved both overall and considering exclusively the switch phase, as confirmed by short TMDs performed starting from the end of the lag phase at different fixed biases ( Fig. 4 and S10) . Given the furin cleavage site insertion/abrogation relevance in all tested S0 molecular contexts, we finally verified whether its influence is length-or sequence-dependent by mutating 681 PRRA 684 into 681 GSAS 684 to construct the RaTG13 GSAS and SARS-CoV-2 GSAS systems. The former 1-up state shows a RBD opening and rotation highly similar to RaTG13 PRRA , suggesting that the conformational changes may be induced by the increased SD2 length (Fig. 4C) . However, SARS-CoV-2 GSAS 1-up RBD aperture is the closest to SARS-CoV-2 RBD , indicating that both SD2 length and a polybasic sequence are needed to compensate for SARS-CoV-2 RBD tendency to open. Altogether, while we cannot precisely determine when the furin cleavage motif developed along SARS-CoV-2 evolution, these results strongly support a progressive onset and a continuous co-evolution of the SD2 domain with the RBD, further optimized by adaptation in all spike domains.

Bats species belonging to different families harbor the highest diversity of α-and βcoronaviruses worldwide and have been identified as the ancestral source of five out of seven CoV species of medical interest (1). Also, CoVs diversity proportionally increases with the number of different bat species co-existing in a single habitat and intra-host receptor polymorphisms (24) . The tight co-evolution of CoVs and bats is particularly evident when SARSr viruses and their long-term natural host -the horseshoe bat R. sinicus -are considered. Indeed, high ACE2 diversity is found exclusively at the RBD-binding interface and different ACE2 alleles sustain to a variable extent bat SARSr RBD binding and entry in a virus species-specific manner, suggesting an intra-host spike evolutionary process driven by the transfer between host subpopulations and subsequent adaptation (15) . The same study also reported that the RBD of two of the closest SARS-CoV-1 ancestors, RsWIV1 and RsSHC014, bound with a 10-fold higher affinity human than R. sinicus ACE2 (10 -7 and 10 -6 M, respectively), while SARS-CoV-1 RBD displayed the same trend but with a 1000-fold difference in favor of hACE2 (10 -8 and 10 -5 M). This demonstrates that, considering exclusively RBD affinity for the receptor, several bat SARSr viruses could in principle infect humans without prior adaptation, but sustained intra-human transmission requires a tighter binding to hACE2. In addition, RBD mutations accumulated along the evolution in palm civets and humans have increased the affinity for their ACE2 at the expense of the efficient binding to that of the reservoir species. Worth mentioning, a similar process has been postulated for MERS-CoV as well, despite different receptor usage, host species tropism and permanence in intermediate hosts (25) . Altogether, RBD/ACE2 affinity measurements of human and ancestor viruses for their respective host receptor efficiently recapitulate their evolutionary trajectory, suggesting this affinity-based strategy can be used to characterize other spillover events.

The intense search of the animal reservoirs that followed SARS-CoV-2 emergence has led to the identification of a novel clade in the Sarbecovirus lineage that comprises viruses infecting Rhinolophus spp. bats and pangolins, including the highly related, ACE2-binding virus RaTG13 in R. affinis bats (5, 7, 8) . While it must be noted that the clade characterization will require more thorough investigations, the close phylogenetic proximity between RaTG13 and SARS-CoV-2 supports the applicability of affinity measurements to trace back SARS-CoV-2 origin. Our SPR measurements of the RBD/ACE2 complexes point out to unique binding features representative of an evolutionary trajectory radically different from that of SARS-CoV-1. Indeed, SARS-CoV-2 and RaTG13 RBDs both have affinities for affiACE2 and hACE2 in the low nanomolar range (10 -8 M) despite sequence divergence.

Compared to SARS-CoV-1, SARS-CoV-2 displays similar binding to hACE2 but a 1000fold higher affinity to its ancestor natural host receptor, while RaTG13, compared to SARSr viruses, has a 100-and 10-fold higher affinity for its natural host receptor and hACE2, respectively. Therefore, considering its RBD affinity for hACE2, RaTG13 has a higher potential of directly crossing the species barrier to humans than previously determined for bat SARSr viruses (26) . Also, the fact that SARS-CoV-2 RBD is endowed with identical binding features and poorly binds to other Rhinolophus spp. ACE2 as recently determined, strongly suggest the virus directly evolved in R. affinis (27) . Altogether, the affinity pattern we 

Origin and evolution of pathogenic coronaviruses

Evidence Supporting a Zoonotic Origin of Human Coronavirus Strain NL63

evolution and classification of bat coronaviruses in the aftermath of SARS

Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics

Clinical characteristics of COVID-19 in 104 people with SARS-CoV-2 infection on the Diamond Princess cruise ship: a retrospective analysis

Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses

A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins

Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science (80-. )

Structural basis of receptor recognition by SARS-CoV-2. Nat

SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects

WHO-convened global study of origins of SARS-CoV-2: China Part

Evolutionary Arms Race between Virus and Host Drives Genetic Diversity in Bat Severe Acute Respiratory Syndrome-Related Coronavirus Spike Genes

WebLogo: A Sequence Logo Generator

Natural Transmission of Bat-like Severe Acute Respiratory Syndrome Coronavirus 2 Without Proline-Arginine-Arginine-Alanine Variants in Coronavirus Disease

The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion

Structural basis for the different states of the spike protein of SARS-CoV-2 in complex with ACE2

Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in

Furin cleavage sites naturally occur in coronaviruses

Global patterns in coronavirus diversity

Bat Origins of MERS-CoV Supported by Bat Coronavirus HKU4 Usage of Human Receptor CD26

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

ACE2 receptor usage reveals variation in susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species

Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2

The molecular basis for SARS-CoV-2 binding to dog ACE2

Cross-species recognition of SARS-CoV-2 to bat ACE2

Altan-Bonnet, N. Altan-Bonnet, β-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway

Emergence of SARS-CoV-2 through recombination and strong purifying selection

The receptor binding domain of SARS-CoV-2 spike protein is the result of an ancestral recombination between the bat-CoV RaTG13 and the pangolin-CoV MP789

SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing

Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension

Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF

How good are my data and what is the resolution?

More and better reference data for improved all-atom structure validation

Towards automated crystallographic structure refinement with phenix.refine

Validation of Structures in the Protein Data Bank

Simmerling, ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB

GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers

MD-TASK: a software suite for analyzing molecular dynamics trajectories

Controlling the SARS-CoV-2 spike glycoprotein conformation

PLUMED 2: New feathers for an old bird

Linking crystallographic model and data quality

Vector pCAGGS Containing the SARS-Related Coronavirus 2

NR-52310; Vector pCAGGS Containing the SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike Glycoprotein Receptor Binding Domain (RBD)

Funding: Research in the Forneris Lab is supported by Fondazione Giovanni Armenise

My First AIRC Grant" id. 20075 to FF), the Mizutani Foundation for Glycoscience (grant id

the NATO Science for Peace and Security Program

None of the funding sources had roles in study design, collection, analysis and interpretation of data

Shown data are the mean of four replicates. (B) Structure comparison of SARS-CoV-2 (PDB ID 6M17) and RaTG13 (reported here) RBD/hACE2 complexes. Whole structures are depicted in ribbon, hACE2, RaTG13 RBD and SARS-CoV-2 RBD are colored in shades of blue, pink and green, respectively. The side chain of RaTG13/SARS-CoV-2 mutations are

We thank Mr. Matteo De Marco for their assistance in molecular cloning and biophysical analyses, and scientists from ARDIS SRL for useful discussions on SPR data processing. We thank Dr. Stefano Iula for useful discussion on geometric measurements. We thank FSTechnology SpA for providing part of the computational resources. We thank the European Synchrotron Radiation Facility (ESRF) for the provision of synchrotron radiation facilities and for the excellent support provided by the ESRF beamline scientists during remote data collection sessions. The following reagents were produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH:

Competing interests: Authors declare that they have no competing interests.Data and materials availability: Coordinates and structure factors for the RaTG13 RBD/hACE2 complex have been deposited in the Protein Data Bank under accession codes 7P8I (crystal form I) and 7P8J (crystal form II).