Fig. 4: Cryo-EM analysis of bioreactor produced PV2-SC6b and PV2-SC5a VLPs.

Gold standard Fourier shell correlation (GSFSC) curves calculated between two independent half sets of data as a function of resolution are shown for PV2-SC6b (A) and PV2-SC5a (B), generated after local resolution analysis in CryoSPARC. Isosurface representations of the electron potential maps for PV2-SC6b (C) and PV2-SC5a (D) are shown at a threshold of 4σ (σ is the standard deviation of the map). Representative fivefold, threefold and twofold symmetry axes are indicated with symbols, and the white triangle delimits an icosahedral asymmetric unit (AU). A central slice through half of each VLP is viewed along the icosahedral twofold axis. Maps are coloured by local resolution (in Å) according to the colour key. Molecular cartoon representations of a single capsid protomer for PV2-SC6b (E) and PV2-SC5a (F) is shown within a triangle depicting an icosahedral AU, with symmetry axes labelled with symbols as in (C) and (D). Individual subunits of the capsid protomer are coloured blue (VP1), green (VP0) and red (VP3). Stabilising mutations engineered into each VLP are shown as spheres coloured magenta and labelled. For VP2 D57A sequence numbering for the mature VP2 peptide is used (equivalent to D126A in VP0 numbering). The sphingosine lipid moiety modelled into the VP1 hydrophobic pocket is shown as sticks coloured orange and labelled (SPH). Close up views of the stabilising mutations and SPH in PV2-SC6b (G) and PV2-SC5a (H) are depicted as sticks fitted in the cryo-EM electron potential maps, shown as a wire mesh (threshold 2σ). SPH for PV2-SC5a is shown fitted into the map at a threshold of 1.5 σ. For PV2-SC6b the mutation in subunit VP4 I57V (I57V in VP0) and for PV2-SC5a the mutation in subunit VP1 T41I, were in disordered regions of the map and were not modelled.