Cryo-electron microscopy structure of the SADS-CoV spike glycoprotein provides insights into an evolution of unique coronavirus spike proteins

Hongxin Guan; Youwang Wang; Abdullah F.U.H. Saeed; Jinyu Li; Syed Sajid Jan; Vanja Perculija; Yu Li; Ping Zhu; Songying Ouyang

Correspondingly in the present investigation of SADS-CoV, we portrayed the 114 general structure of the S protein and the organization of its structural (Fig. 1A) . The size-exclusion chromatography (SEC) result showed that the 138 protein sample is a trimer in the solution and the purity of more than 95% 139 examined in SDS-PAGE analysis (Fig. S1A, S1B) . The protein displayed high 140 homogeneity in cryo-EM screening and was then diluted to 0.63 mg ml -1 for 141 the data collection. Cryo-EM micrograph movies were collected on a Gatan 142 K2 direct electron detector mounted on an FEI Titan Krios electron 143 microscope (Fig. S2) . Following reference-free 2D classification of the S 144 protein, we determined a three-dimensional (3D) structure of the SADS-CoV 145 S trimer at 3.55 Ã… resolution judged by the gold-standard Fourier shell 146 correlation (FSC) criterion of 0.143 (Fig. 1B, 1C and Table 1 ). The resolved 147 atomic structure of prefusion SADS-CoV S ectodomain covers almost all of 148 the key structural elements, as shown in Fig. 1A except residues 81-101 of 149 NTD and residues 999-1068 of HR-C (Fig. 1D, Fig. S3 ). Forty-five (15 on The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.04.976258 doi: bioRxiv preprint stabilized by 30 pairs (10 on each subunit) of disulfide bonds (Fig. S4) . The S 153 trimer shows a dome-like shape, with three S1 heads forming a crown-like 154 structure and sitting on top of a trimeric S2 stalk (Fig. 1D, 1E , and Movie S1). 155 The trimer spike has a length of 143.2 Ã… from S1 to S2 and a width of 111.3 Ã… 156 and 61.4 Ã… at S1 and S2, respectively (Fig. 1E) . Three S1-CTDs are located 157 at the top center of the S trimer arranged as a small triangle, whereas three 158 S1-NTDs are located on the lower and outer side of S1-CTDs arranged as a 159 big triangle. This architecture, i.e., CTD sandwiched by its own NTD and the 160 adjacent NTD, comes into being one side of the big triangle (Fig. 1F) . The 161 cleavage site of S1 and S2 subunits locates between residues 544 (Val) and 162 545 (Arg). The central helicase N (CH-N) and C (CH-C) of S2 from each 163 subunit form a six-helix bundle in the core of S trimer. Heptad repeats N (HR-164 N) contains four helices connected by three loops locked between the S1 and 165 S2 subunit at the outside of the S2 stalk, while part of the HR-C is missed in 166 the structure (Fig. 1G) . Each monomeric subunit of S1 contains two major 167 domains, S1-NTD and S1-CTD which are used to bind attachment sialic acid 168 receptors or protein receptors and play a vital role in the CoV entry, and two 169 subdomains, SD1 and SD2 whose function are not very clear ( Fig. 1H-1I , 170 Movie S1).