Extended Data Fig. 1: Purification of HMPV-L:P and structure determination using cryo-EM.

a, Representative size-exclusion chromatogram of the L:P complex (these experiments were repeated more than five times). Fractions indicated by an arrow were collected and concentrated to 0.85 mg ml⁻¹ and used for cryo-EM analysis. Inset, SDS–PAGE followed by Coomassie blue staining of the purified samples. Free P protein separated from the L:P complex by heparin chromatography is also shown. For gel source data, see Supplementary Fig. 1. b, Raw micrograph of HMPV-L:P particles recorded in vitreous ice. Scale bar, 10 nm. c, Power spectrum of the image shown in b. We limited the high resolution for fitting to a spatial frequency of 1/5.0 Å, and 1/2.9 Å marks the highest spacing to which CTF rings were successfully fit. d, Two-dimensional classes and ‘self-consistency check’ for the cryo-EM three-dimensional reconstruction. For each pair of boxes (top and bottom) across the three rows, the top image shows one two-dimensional class average and the bottom image shows the corresponding projection from the initial three-dimensional model. e, Local resolution of the cryo-EM density map. Variations in local resolution are colour-coded from blue (3.0 Å) to red (5.9 Å), computed with Resmap⁴⁴. f, FSC of the cryo-EM map as a function of the spatial frequency. The gold-standard resolution is 3.7 Å based on the FSC = 0.143 criterion, consistent with the model-to-map correlation (0.5 criterion). g, Example of the electron-density map that enabled model building. The region shown is at an interface between the RdRp and capping domain. The map is shown as a grey mesh, contoured at a level of 3σ. The atomic model is shown as sticks with residues from RdRp coloured in cyan (NTD in grey) and in green for the capping domain. h, The region shown is the three-stranded β-sheet at the interface between the RdRp (cyan sticks) and the phosphoprotein (magenta sticks). The map is shown as a grey mesh, contoured at a level of 2.5σ. We observed a nearly identical structure of the L:P complex in a reconstruction obtained by premixing the L:P complex with fully phosphorylated P, indicating that potential exchange of P affected neither the formation nor the structure of the L:P complex.