Superdex 200 column

The globular head of H5 HA was expressed using the Bac-to-Bac baculovirus expression system (Invitrogen). The DNA fragment corresponding to residues D55 and E271 (F55-271) of the A/Anhui/1/2005 HA was inserted into a pFastBac dual vector (Invitrogen), with an N-terminal gp67 signal peptide to facilitate secretion and a C-terminal 6-His tag for purification. The recombinant bacmids were transfected into Sf9 insect cells using Cellfectin II Reagent (Invitrogen). Secreted globular head protein in the cell supernatants was captured by nickel-nitrilotriacetic acid (Ni-NTA) resin, eluted with 500 mM imidazole, and then further purified by size exclusion chromatography on a Superdex 200 column (GE Health Care). The Fab fragment was produced by digesting full-length 65C6, 100F4 and AVFluIgG03 IgG with papain for 5 h using Pierce Fab Preparation Kit (Thermo #44985) and further purified by gel filtration (Superdex 200 column, GE Health Care). The globular head and Fab complexes were generated by mixing the two components together, incubated on ice for 1 h and purified by size exclusion chromatography on a Superdex 200 column (GE Health Care).

Plasmids encoding prefusion RSV F (DS-Cav1), L141W, and D489Y variants and post-fusion (F ΔFP) proteins based on strain A2 were transfected into FreeStyle 293 F and Expi293 cells, respectively (Invitrogen, Carlsbad, CA, USA)^{32 (link), 34 (link)}. Proteins were expressed in the presence of kifunensine (5 μM) and were purified over Strep-Tactin resin (IBA, Goettingen, Germany). Prefusion RSV F (PRDM) protein^{36 (link)} for DSF studies was purified from transiently transfected Expi293 cells using a two-step purification protocol including cation-exchange chromatography at pH 5.0 (HiTrap Capto SP ImpRes column; GE Healthcare Biosciences, Pittsburgh, PA, USA) and size-exclusion chromatography using a Superdex 200 column (GE Healthcare). For crystallization studies, purification tags were removed by overnight digestion with thrombin followed by gel filtration using a Superose 6 column (GE Healthcare Biosciences) with a running buffer of 2 mM TRIS pH 8.0, 200 mM NaCl. For ITC studies, tags were not removed and proteins were purified by gel filtration using a Superdex 200 column (GE Healthcare) with a running buffer of PBS.

The purified FLD21.140 IgG was digested using papain from papaya latex (P3125; Sigma) at 37 °C for 12 h, and the digested Fab and Fc fragments were separated by loading the mixture onto a protein A column. The Fab fragment was collected in the flow-through, and the Fc fragment was captured on the protein A beads. The flow-through was further purified by gel filtration (Superdex 200 column, GE Healthcare). The purified globular heads (A/Thailand/1(KAN-1)/2004) and FLD21.140 Fab were mixed at a molar ratio of 1:1, incubated at 4 °C for 1 h, and then purified by gel-filtration chromatography on a Superdex 200 column (GE Healthcare). For crystallization, the globular head–FLD21.140 Fab complex was concentrated to 20 mg/ml in HBS buffer. Crystals were successfully grown at 18 °C using the vapor diffusion method in sitting drops, which consisted of equal volumes of protein and reservoir solution containing 2.0 m (NH₄)₂SO₄ and 0.1 m BisTris, pH 5.5. All crystals were cryoprotected by soaking in reservoir solution with 20% v/v glycerol for several seconds and then frozen in liquid nitrogen before data collection. Diffraction data were collected on the BL17U beamline at the Shanghai Synchrotron Research Facility (SSRF) (65 ) and processed using the program HKL2000 (66 (link)). All data collection and processing statistics are listed in Table S2.