The emergence of SARS-CoV-2 in late 2019 has led to a global pandemic and has 29 disrupted lives and global economies on a scale unseen in recent human history. 30 This is not the first time when a new coronavirus has posted as a major threat to 31 public health; both SARS-CoV and Middle East Respiratory Syndrome (MERS-CoV) 32 caused human infections within past 17 years (1) . The fact that no licensed vaccines 33 have ever been approved for these highly similar viruses is a reminder for the great 34 challenges we face when hundreds of companies and institutions worldwide rush to 35 develop COVID-19 vaccines with multiple strategies (2) . 36 A successful vaccine that could truly impact the course of this ongoing COVID-19 37 pandemic has to have four key characteristics: safety, efficacy, scalability (to billions 38 of doses to meet global demand), and speed. Although protein subunit vaccines have 39 excellent track records for the first three requirements, exemplified by the highly 40 successful vaccine Gardasil used to prevent HPV infections (3) and Shingrix vaccine 41 for containing herpes zoster virus infections (4), subunit vaccine development can 42 take years to decades to complete. Many of the difficulties reside in the 43 manufacturing processes that have to ensure a fully native-like antigen structure is 44 retained, starting from subunit vaccine designs. Similar to other enveloped RNA 45 viruses such as HIV, RSV and influenza, coronaviruses including SARS-CoV-2 also 46 use a ubiquitous trimeric viral surface antigen (Spike protein) to gain entry into host 47 of oleic acid seen in our structure, linoleic acid as well as PS80 were also observed 118 in the recently published structure of a full-length mutant S protein (3Q-2P-FL) 119 produced from insect cells (7) . PS80 is buried deeply in the hydrophobic pocket 120 residues with a few hydrophilic residues including N99, N121, R190 and H207 121 making hydrogen bonds with the hydroxyl group of the PS80 (Fig. 2B) . Notably, PS80 122 engages hydrophobic interactions with F175 and M177 which are invisible in the S-123 2P structure (Fig. 2C) . Since no small molecule was shown to bind to the S-2P 124 structure, PS80 likely stabilizes the disordered loops of the NTD domain, making 125 them more ordered (Fig. 2B and 2C ). The oleic acid located in the hydrophobic 126 pocket of the RBD domain engaged a salt bridge interaction with R408 at the 127 adjacent protomer through its carboxylic acid group, bringing the RBD domain in 128 close proximity and resulting in the tightly closed conformation (Fig. 2D) . 129
Recent studies have shown that low pH can stabilize the S-Trimer (12). Indeed, 131 negative staining EM analysis of WT S-Trimer at pH 5.5 revealed more homogenous 132 trimer than that at physiological pH (See accompanying paper). In light of this finding, 133 we were able to determine the cryo-EM structure of the WT S-Trimer at 3.2 Å 134 resolution at pH 5.5 ( Fig. S7 and Table S1 ). The structure of the WT S-Trimer 135 resembled that of the MT form with a root mean square deviation of 0.5 Å over 2773 136
Cα atoms (Fig. 3A) . Oleic acid was well resolved in the WT structure but the density 137 for the PS80 was weak, likely due to the low resolution or low occupancy. As a result, 138 the NTD domain of the WT S-Trimer was less well resolved than that of MT (Fig. 3A) . 139
It has been shown that a pH-dependent switch domain (residue 824-848, pH switch 140 1) undergoes dramatic conformational change at different pH values (12). However, 141 this region was nearly identical between our two structures (Fig. 3B ). Instead, a 142 fragment (residue 617-639) we named pH switch 2 at the CTD1 region of the S1 143 domain before the furin cleavage site displays significant structural arrangement. 144 to this structural arrangement. At physiological pH, R319 forms salt bridge 148 interactions with D737 and D745 (Fig. 3D) . At lower pH, the protonation of D737 and 149 D745 weakens these interactions. As a result, R319 flips to the other side and makes 150 hydrophobic interactions with W633 and L629 through its aliphatic chain, leading to 151 the ordered helix-turn-helix motif (Fig. 3E) . The newly-formed structural motif makes 152 direct contact with the previously identified pH switch 1 of the adjacent protomer, 153 accounting for the enhanced stability of the WT S-Trimer at lower pH (Fig. 3A) . The 154 structural arrangement of pH switch 2 in different pH was also observed in previous 155 studies (12), further supporting the conformational change between the MT and WT 156 S-Trimer structures was due to the different pH but not to the mutation in the furin 157 site. 158
In contrast to the structural differences described above for S-2P protein, both of our 160 WT and MT S-Trimer were nearly identical to the recently published structures of full-161 length wild-type S (10) and 3Q-2P-FL (7) purified in detergent from HEK293 and sf9 162 insect cell membranes, respectively. When revisiting the electron density map for full-163 length wild-type S protein (EMDB: 22292), we spotted unassigned density at the become predominant over the ancestral form worldwide and has been shown to interaction with K854 at the conformational switch region (Fig. 4C ). From the tightly to 178 the loosely closed state, the conformational switch undergoes a large conformational 179 arrangement and becomes disordered (Fig. 4D ). K854 flips to the opposite side and 180 interacts with D568 and D574 of the CTD1, causing the S1 to move downwards 181 relative to the S2 (Fig. 4A) . Finally, the CTD1 domain further moves downwards and 182 causes the RBD to adopt an open conformation for receptor binding (Movie S1). Like the previously reported structure of full-length WT S protein purified in detergent 199 micelles, it is unclear whether the furin cleavage site in our resolved WT S-Trimer 200 structure is cleaved. Moreover, we could not exclude the possibility that other 201 conformational states exist in the WT sample that were not captured in our cryo-EM 202 study since partial cleavage of the furin site may lead to some S1 dissociation from 203 S-Trimer. Nevertheless, we are certain that the highly purified WT S-Trimer 204 predominately adopts a pre-fusion state, unlike the full-length wild-type spike protein 205 which forms both pre-and post-fusion states in the presence of detergent (10). We thank Xiaodong Wang for his coordination and input in this study. We thank 311
Maofu Liao and Andrew C. Kruse for critical reading of the manuscript. We thank 312
Hongwei Wang for providing graphene oxide coated grids. We also thank staff at 313 Shuimu BioSciences for their assistance with cryo-EM data collection. All EM data 314 were collected at Shuimu BioSciences. Cell culture medium was clarified by depth filtration (Millipore) to remove cell and debris. S-Trimers were purified to homogeneity by consecutive chromatographic steps including Protein A affinity column using MabSelect PrismA (GE Healthcare) which was preloaded with Endo180-Fc at (3 mg/mL) to capture S-Trimer, based on the high affinity binding between Endo180 and Trimer-Tag (2) . After washing off any unbound contaminating proteins, S-Trimers were purified to near homogeneity in a single step using 0.5 M NaCl in phosphate buffered saline (PBS). For S MT and SARS-CoV S-Trimer, the proteins were dialyzed against PBS plus 0.02% Polysorbate 80 before analysis. After one hour of low pH (pH 3.5) viral inactivation (VI) step using acetic acid, the pH was adjusted to neutral range, WT S-Trimer was further purified on a Capto QXP resins (GE BioSceinces) in a flow-through mode to remove any host cell DNA and residual host cell proteins (HCP). A final preventative viral removal (VR) step was performed using a nano-filtration cartridge (AsahiKASEI) before final buffer exchange to PBS plus 0.02% Polysorbate 80 by UF/DF (Millipore).
ACE2-Fc expression vector was generated by subcloning a gene-synthesized cDNA template (GenScript) encoding soluble human ACE2 (amino acid residue 1-738，accession number: NM_001371415.1) into
Hind III and Bgl II sites of pGH-hFc expression vector (GenHunter, Nashville, TN) to allow in-frame fusion to human IgG Fc. The expression vector was then stably transfected into GH-CHO (dhfr -/-) cell line and high expression clones were selected and adapted to SFM-4-CHO (Hyclone) serum free medium and ACE2-Fc was produced in a 15 L bioreactor as essentially as described for S-Trimer above. ACE2-Fc was purified to homogeneity from the conditioned medium using PoRos XQ column (Thermo Fisher) following manufacturer's instructions.
The avidity of different S-Trimer binding to the SARS-CoV-2 receptor ACE2 were assessed by Bio-Layer Interferometry measurements on ForteBio Octet QKe (Pall, New York). ACE2-Fc (10 µg/mL) was immobilized on Protein A (ProA) biosensors (Pall). Real-time receptor binding curves were obtained by applying the sensor in a two-fold serial dilutions of S-Trimer from 22.5-36 µg/mL in PBS. Kinetic parameters (K on and K off ) and affinities (K D ) were analyzed using Octet software, version 12.0.
Dissociation constants (K D ) were determined using steady state analysis, assuming a 1:1 binding model for a S-Trimer to ACE2-Fc.
Negative staining was performed as previously described (3) . In brief, 3 μl of purified S-trimer at a concentration of about 0.01 mg/ml was deposited on a glow-discharged carbon-coated copper grid for 30 s before being blotted with filter paper. Grids were quickly washed with two drops of water and one drop of 2% (w/v) uranium acetate. Grids were kept touching to the last drop of 2% (w/v) uranium acetate for 90 s, and blotted with filter paper. Data collection was performed on a Tecnai T12 electron microscope operated at 120 Kev equipped with a FEI Ceta 4K detector. Images were collected at a magnification of 57,000 x and a defocus of 1.5 μm.
Purified MT S-trimer protein diluted to 0.2 and 0.5 mg/ml in PBS buffer were applied to glow-discharged gold holey carbon 1.2/1.3 300-mesh grids with and without graphene oxide, respectively. Grids were blotted for 2-4 seconds at a blotting force of 4 and plunge-frozen in liquid ethane using a MarkIV Vitrobot (Thermo Fisher Scientific). The chamber was maintained at 8 ºC and 100% humidity during freezing. 0.3 mg/ml of WT S-trimer sample in low pH buffer (100 mM sodium citrate pH 5.5 and 100 mM NaCl) was deposited on glow-discharged gold holey carbon 1.2/1.3 300-mesh grids with graphene oxide. Grids were blotted and vitrified using the same condition.
All movies were collected using a Titan Krios microscope (Thermo fisher Scientific) equipped with a BioQuantum GIF/K3 direct electron detector (Gatan). The detector is operated in superresolution mode.
A complete description of cryo-EM data collection parameters are summarized in Table S1 .
For MT S-Trimer protein, motion correction for cryo-EM images and contrast transfer function (CTF) estimation were performed using motioncorr2 (4) and CTFFIND4 (5) respectively. 574,832 particles were automatically picked from 534 images collected on grid without graphene oxide (GO) using Laplacian-of-Gaussian in Relion 3.0.7 (6), and 1,029,938 particles was automatically picked from 1584 images collected on grid with GO. Extract particles from two datasets were downsized by two-fold and subjected to 2D classification separately, resulting in 438,205 and 560,545 good particles. Particles from GO grids were recentered using scripts written by Kai Zhang (http://www.mrclmb.cam.ac.uk/kzhang/useful_tools/scripts/) before 3D classification. Good particles from both datasets were combined and subjected to 3D classification using the initial model generated from the model of S protein with C1 symmetry (PDB ID: 6vxx). Two major classes accounting for 26.7% and 25.8% particles show clear and complete structural features. These particles were auto-refined followed by local 3D classification with C1 symmetry to generate two classes with similar structure. The better class was subjected to 3D refinement with C3 symmetry and post-processed using mask on the entire molecule to yield a 2.9 Å map. CTF refinement was performed to further increase the resolution to 2.6 Å.
For image processing of WT S-Trimer, MT S-Trimer map was low-pass-filtered to 20 Å resolution and used as the 3D reference template for auto-picking. 752,204 articles were picked from 1199 images collected on GO-coated grid and used for two rounds of 2D classification. 541,528 particles were selected from good 2D classes and used for 3D classification with C1 symmetry. One class accounting for 32.2%
showing a well-defined structure was refined with C3 symmetry and post-processed using mask on the entire molecule to give a map at 3.4 Å resolution. CTF refinement followed by another round of 3D refinement improved the resolution to 3.2 Å. Reported resolutions were calculated based on the goldstandard Fourier shell correlation (FSC) at the 0.143 criterion.
The soluble ectodomain structure S-2P (PDB: 6VXX) was used as a template for model building. The missing region in the S-2P structure can be de novo modeled in the MT S-Trimer map, owing to its high resolution. The model was manually built in COOT (7) and real space refinement was performed in Phenix (8) The relative quantitation for oleic acid and linoleic acid was performed by a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer. The chromatographic separation was performed using a Waters CSH C18 column (2.1x100 mm, 1.7 μm) and maintained at 40°C. The separation was performed using isocratic flow of a solvent composed of 90% acetonitrile, 10% water, and 2 mM ammonium acetate. The flow rate was set at 0.2 mL/min for 10 min.
Full-scan-ddMS 2 mass spectra were acquired in the range of 100-1500 m/z with the following ESI source settings: spray voltage 2.5 kV, aux gas heater temperature 380 °C, capillary temperature 320 °C, sheath gas flow rate 30 unit, aux gas flow gas 10 unit in the negative mode. 