Ultracon concentrator

For the ACE2-bound or S309-bound Omicron spike and ACE2-bound Omicron spike with L371S, P373S, and F375S mutations, the spike protein was incubated with the purified hACE2 at a 1:4 molar ratio (spike trimer to hACE2) overnight at 4 °C before purification by concentration and dialysis using a 100-kDa cut-off Ultracon concentrator (Millipore) with 20 mM Tris (pH 8.0) and 150 mM NaCl. For the Omicron RBD-hACE2-S304 complex, RBD and hACE2 were co-incubated for 2 h at 4 °C at a 1:1.2 molar ratio. The mixture was then purified via gel filtration using a Superdex^TM 200 10/300 GL (GE Healthcare) to obtain the Omicron RRD-hACE2 complex. The Omicron RBD-hACE2 and S304 Fab were then mixed at a 1:1.5 molar ratio and incubated overnight at 4 °C, followed by gel filtration using a Superdex^TM 200 10/300 GL (GE Healthcare) to obtain Omicron RBD-hACE2-S304 complex. The protein complex was stored in a buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl for cryo-EM sample preparation.

Purified hACE2 was mixed and incubated with BF.7 RBD, XBB RBD, XBB.1.5 RBD, BQ.1 RBD and BQ.1.1 RBD at a 1:1.5 molar ratio (hACE2 to RBD) on ice for about 2 h. The six mixtures were then purified on Superdex™ 200 10/300 GL column (GE Healthcare) in a buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl. The XBB.1.5 RBD/hACE2 was mixed with S304 Fab at a 1:1.5 molar ratio (hACE2-RBD to Fab) on ice for about 2 h and purified on Superdex™ 200 10/300 GL column (GE Healthcare) with 20 mM Tris (pH 8.0) and 150 mM NaCl. Purified complex proteins (BF.7 RBD/hACE2, XBB RBD/hACE2, XBB.1.5 RBD/hACE2/S304 Fab, BQ.1 RBD/hACE2 and BQ.1.1 RBD/hACE2) were concentrated to 2 mg/mL for cryo-EM using a 30-kDa cut-off Ultracon concentrator (Millipore).