Quikchange protocol

Site-specific cysteine replacements were introduced into YF1 via the QuikChange protocol (Invitrogen, Life Technologies) within both the pET-41a expression construct and the pDusk-myc-DsRed reporter plasmid^{19 (link)}. Light-dependent signal transduction of YF1 variants was assessed in the pDusk reporter context^{19 (link)}. YF1 variants were expressed in CmpX13 cells^{42 (link)} and purified as described^{19 (link)}. Labelling was conducted with 10-fold molar excess of MTS. Light-dependent catalysis of MTS-labelled YF1 variants was assessed in vitro as described^{12 (link)}. For EPR measurements, buffer was exchanged to deuterated HEPES supplemented with 50% (v/v) per-deuterated glycerol. Samples were kept in darkness or were illuminated for 5 min at 450 nm and were rapidly cooled in liquid nitrogen. Pulsed ELDOR spectra were recorded using the DEER sequence^{22 (link), 23 (link)} Experiments were performed at 40 K in X- and 50 K in Q-band. ELDOR data were evaluated using DeerAnalysis^{43 (link)}. Label rotamer simulations were performed using MMM^{44 (link)} and MtsslWizard^{45 (link)}. ENM was performed in MMM, RBD in mtsslDock^{26 (link)}. Molecular graphics were prepared with PyMOL⁴⁶ . For details, see SI.

RNA of H7N7 viruses was extracted using QIAamp Viral RNA Mini Kit (Qiagen). Recombinant viruses were generated using reverse genetics. For this purpose, all gene segments of each virus were amplified and cloned into the plasmid pHWSccdB as previously described45 (link). Introduction of the selected mutations into the HA gene segments of different LP and HP viruses in this study was done using site-directed mutagenesis (primer sequences are available upon request) following the QuikChange protocol (Invitrogen). All recombinant viruses and mutants were rescued in HEK 293 T/MDCKII co-culture45 (link). Supernatants were then inoculated in SPF ECE for 3–5 days. Eggs were candled daily and those with dead embryos were kept in 4 °C and allantoic fluid was collected. Egg fluids with HA titre >16 (4 log₂) were used for further investigations. To exclude any unwanted mutation and confirm the introduced genetic changes in the constructed viruses, viral RNA was extracted and analysed by Sanger sequencing of RT-PCR amplicons as described46 (link).