Fig. 3. Structural characterization of AAV9 interaction with human IL3.

a Schematic depicting intermolecular cross-links between human IL3 (hIL3) and AAV9 with XlinkX scores above 40, indicating high confidence⁸³. b Structure of AAV9 (PDB ID: 3UX1) trimer indicating human IL3 cross-linking amino acids (red) and the capsid variable regions (VR) within reach of the cross-linker (VR-I: pink, VR-V: purple, VR-VI: teal, VR-VII: cyan, VR-VIII: blue, VR-IX: green). c SPR of human IL3 binding for AAV9 and chimeric capsids containing AAV8 amino acid identity at the indicated variable regions. v.g.: viral genomes. d Left: A central slice from a cryo-electron tomogram of AAV9 with human IL3. Arrows in magnification indicate AAV9 capsids. 46 3D tomograms, composed of 41 such images each, yielded 85,135 symmetry-expanded particles for sub-tomogram averaging. Middle: Map of AAV9 trimer bound by human IL3-Fc obtained after symmetry expansion and particle subtraction followed by multiple rounds of refinement. Human IL3-Fc density is segmented in red. Right: The same map overlaid with a model of the AAV9 trimer (PDB ID: 3UX1) highlighting the variable regions relevant for human IL3 binding (VR-I: pink, VR-V: purple).