Figure 2. Structure of HsFpn bound to Ca²⁺.

(a) Cryo-EM map of HsFpn in complex with 11F9 in the presence of Ca²⁺. Densities for NTD, CTD, and 11F9 are colored in pale green, light orange, and slate purple, respectively. A smoothened map contoured at a low threshold (translucent grey) is overlaid to show the lipid nanodisc density around Fpn. (b) Structure of HsFpn with a bound Ca²⁺ in an outward-facing conformation. NTD and CTD are colored as described in (a). Ca²⁺ is shown as a green sphere. (c) Zoomed-in view of the Ca²⁺ binding site in the NTD. The five residues coordinating Ca²⁺ are labeled and shown as side chain sticks. The density for Ca²⁺ is contoured at 7.5σ as blue mesh. (d) Structural comparison of apo (grey, PDB ID 6W4S), Co²⁺-bound (pink, PDB ID 8DL8), and Ca²⁺-bound (green) HsFpn near S1. The side chains of D39 and H43 are shown as sticks. Co²⁺ is shown as a pink sphere. (e), (f), and (g) Three views of conformational changes in NTD induced by Ca²⁺ binding. The Co²⁺-bound (pink) and Ca²⁺-bound (green) structures are aligned and shown as cartoon with cylindrical helices. (e) Top view (from the extracellular side) of the alignment. The helical directions of TM1-6 are visualized by vectors inside cylinders, and the bending of these helices is indicated by black arrows. Bending of TMs viewed from the front (f) and the back (g). The displacement of the extracellular loop between TM3 and TM4 is marked with a black arrow and distance.

Figure 2—figure supplement 1. Cryo-EM analysis of HsFpn-Ca²⁺ in nanodisc.

(a) Representative electron micrograph (upper panel) and 2D class averages (lower panel). (b) Workflow of single-particle data processing. (c) The gold-standard Fourier shell correlation (FSC) curves (left panel) and map-to-model FSC curves (right panel). (d) Direction distribution of particles used in the final 3D reconstruction. (e) Local resolution map.

Figure 2—figure supplement 2. Cryo-EM densities of TM helices and amphipathic helices (AH).

Densities for HsFpn-Ca²⁺ are shown as green mesh. Residues within the ranges indicated below are rendered in stick representation and colored in pale green for NTD or light orange for CTD.

Figure 2—figure supplement 3. Structural comparisons of Ca²⁺-bound HsFpn.

(a) Differences in Ca²⁺ coordination by HsFpn (green) and by BbFpn (deep blue, PDB ID 6BTX). (b) Comparison of Ca²⁺-bound (green) versus hepcidin-bound (deep purple, PDB ID 6WBV) structure of HsFpn shows further outward movements of NTD. Displacement of the loop between TM3 and TM4 is marked with an arrow and distance. Co²⁺ ions in the Fpn-hepcidin structure are shown as deep purple spheres.