Dh10bac competent cells

The nucleotide sequence encoding VP1 of HuSaV GII.1 (sample ID 18003) was sequenced and cloned into the pFastBac1 vector (Invitrogen, Waltham, MA, USA). Each construct transformed DH10Bac competent cells (Invitrogen) and recombinant bacmid was purified. Recombinant baculovirus production was obtained by transfection of recombinant bacmid into ExpiSf9 cells using the ExpiSf Expression system (Thermo Fisher Scientific) and collecting the supernatant on day 5–6 of infection. Inoculating ExpiSf9 cells with this viral stock, the culture supernatant was harvested to confirm expression. The supernatant was centrifuged at 12,000× g for 10 min, and after repeated centrifugation, the supernatant was passed through a 0.45 μm filter (Merk Millipore, Burlington, MA, USA). After centrifugation at 113,000× g for 4 h, the precipitate was suspended in TNE buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA [pH 7.4]) and left at 4 °C overnight. The samples were then suspended in 42% CsCl-TNE buffer and subjected to cesium chloride equilibrium density centrifugation at 139,000× g for 20 h, after which each fraction was collected and dialyzed against TNE buffer. The final sample was negatively stained with 1% uranyl acetate solution, and the VLP structure was confirmed by transmission electron microscopy (100 kV, JEM-1400, JEOL, Tokyo, Japan).

Recombinant baculovirus was produced using the pFastBac-pfmsp10 vector, DH10Bac competent cells (Invitrogen) and a Spodoptera frugiperda (Sf9) cell line (Invitrogen), following published instructions [16 ]. The first generation of the recombinant baculovirus was designated as P0, and then baculoviruses were passaged two times to obtain a new generation called P2.

The S glycoprotein ectodomain was expressed and purified using a previously described protocol^{17 (link)}. The construct was transformed into bacterial DH10Bac competent cells (Invitrogen); then, the extracted Bacmid was transfected into Sf9 cells (source: American Type Culture Collection) using Cellfectin II Reagent (Invitrogen). The passage 1 (P1) baculoviruses were harvested and amplified to generate a high-titer virus stock with Sf9 cells, and then they were used to produce the recombinant proteins. The supernatant of the cell culture containing the secreted S glycoprotein was harvested at 60 h after infection and concentrated, and the buffer was changed to binding buffer (10 mM HEPES, pH 7.2, 500 mM NaCl). Finally, S glycoprotein was captured by StrepTactin Sepharose High Performance (GE Healthcare) and eluted with 10 mM D-desthiobiotin in binding buffer. Oligomerization of the HCoV-229E S trimer (1 mg) was analyzed using a Superose 6 Increase 10/300 GL column (GE Healthcare) with a buffer containing 10 mM HEPES, pH 7.2, and 150 mM NaCl^{23 (link)} at a flow rate of 0.3 ml/min (4 °C). We found three peaks in the gel filtration profile (Supplementary Fig. 1a). Because peak 1 represents a highly aggregated state, we collected the fractions of peaks 2 and 3 for the cryo-EM analysis. The proteins collected in the different fractions were analyzed by SDS-PAGE.