Fig. 4: Fluad structural analysis by cryo-electron tomography and molecular modeling.

a, b Examples of slices through cryo-electron tomograms with a field of Fluad spike complexes and putative adjuvant vesicles. Black arrows indicate some spike complexes protruding from an apparent lipid bilayer. Black asterisks denote adjuvant vesicles. c Oblique view of a cryoelectron-tomographic 3D reconstruction (gray) of a Fluad large particle. A disc-like structure was observed with spiked densities around its perimeter, which were used to dock HA molecules (ectodomain trimers, PDB ID 3LZG) that were parallel and anti-parallel on either the same or different sides of the disk, respectively. HA heads were colored red, and HA stems were colored blue. d Side-view of the Fluad particle in panel c illustrates parallel and anti-parallel HA orientation. e Side view of a subtomogram average of Fluad disc structures, focusing on the disc center. Two layers of density are visible in a density slice bisecting the disc, which indicated the discs are composed of a bilayer. The distance between the centers of each band of density were 48 Å. f A schematic of the HA trimers on the perimeter of the lipid-based disc (gray) illustrates their consensus orientations with HA heads, colored in red, aligned normal to the disc surface. HA stem regions are colored blue. g Distance and angular measurements between HA-trimers that were docked into Fluad discs. The plot is segmented into colored regions according to Monte Carlo simulations of HA-stem clashes: no steric clash (blue), all steric clash (yellow), and conditional steric clash depending upon the interdigitation of Fabs (green). h HA-trimer distances between the central axis of head domains were tabulated and plotted as a histogram. The arrow denotes the threshold distance of 16 nm under which bivalent binding is possible. Scale bars are 100 nm in panels a and b, and 10 nm in panels c, d, and e. Fluad cryo samples were observed at least three times across multiple microscopes, with morphology of discs being consistent.