Extended Data Fig. 3. Single-particle cryo-EM analysis of human H3N2 FluPol_A bound to cRNA promoter.

a, Representative micrograph of cRNA-bound FluPol_A heterotrimer particles embedded in vitreous ice. b, Representative 2D class averages. c, FSC curves for 3D reconstruction using gold-standard refinement in RELION, indicating overall map resolution of 4.07 Å and the model-to-map FSC. Curves are shown for phase randomisation, unmasked, masked and phase-randomisation-corrected masked maps. d, 3D reconstruction locally filtered and coloured according to RELION local resolution. e, Angular distribution of particle projections with the cryo-EM map shown in grey. f, Cryo-EM density of the PA loop 352-356 at the dimer interface. g, Cryo-EM map of cRNA-bound FluPol_A dimer refined without symmetry imposed (C1), revealing an extra density (green) located next to the 3ʹ end of the 5ʹ cRNA close to the template entry channel. h, Close-up views highlighting cryo-EM extra density (dark green) with the 3′ vRNA strand from the superimposed FluPol_B structure⁵¹ (PDB: 5MSG, light green) inserting into the polymerase active site. Localisation of the 3ʹ vRNA shows that bases are positioned in the extra density facing the density corresponding to the 3ʹ end of the 5ʹ cRNA, suggesting the presence of a promoter RNA duplex region as observed in vRNA-bound FluPol_B ⁵¹. The extra density is consistent with the presence of a 3ʹ cRNA in one of the heterotrimers of the cRNA-bound FluPol_A dimer, oriented towards the polymerase active site.