Fig. 1: FD-based AFM mapping of C5aR receptors and probing their orientation within the lipid bilayer.

a Ribbon diagrams of human C5aR and C5a structures. The interaction between C5a and C5aR is stabilized by two orthosteric binding sites at the receptor extracellular side: a binding site at the N-terminus (shaded in blue) and a functionally important effector site at the extracellular region (shaded in red) of C5aR. The peptide antagonist PMX53 establishes hydrogen bonds with ECL2 at the effector site. The Arg (R) residue at the C-terminal of the C5a ligand is thought to play a key role in stabilizing the interaction with C5aR. b Orientation of lipid bilayer-embedded C5aR is random: they can adopt two orientations, with the intracellular C-terminal His₆-tag facing the inner or the outer side of the lipid bilayer. c Overview AFM topography image (height map) of C5aR reconstituted in liposomes and adsorbed on freshly cleaved mica. Sparsely distributed C5aR particles can be observed protruding from the liposomes. The image was acquired with a bare AFM tip. d Cross-section (white dashed line in inset) showing a C5aR particle protruding 1.7 nm from the lipid bilayer having a diameter of 16 nm. The diameter was measured as full-width at half-maximum (FWHM). Inset: expanded view of a single C5aR particle. e 2D histogram of height and diameter of C5aR receptors imaged in (c). The diameter distribution shows three main populations, while the height distribution shows two main peaks. Data in (c) and (e) are representative of at least five independent experiments.