Supplementary Figure 2: Zn²⁺ binding and S4 position

(a–d) Se–Met and Zn²⁺ anomalous Bijvoet signals. These blue mesh show the initial map phasing by Se–MAD at 4.3Å (contoured at 1.0σ level). All Bijvoet anomalous difference signals (green mesh, contoured at 3.0σ level) were calculated by Se–Met1(a), Se–Met2(b), and Se–Met3(c) data collections, respectively. The asterisk shows unknown weak Bijvoet anomalous signal. The red mesh shows the Bijvoet anomalous difference map of Zn²⁺ calculated by Cryst–Z1 data collection (contoured at 5.0σ level) (d). (e) Electron density map of mHv1cc and anomalous Bijvoet signal of Zn²⁺, shown in stereogram (wall-eyed viewing). σ_A–weighted 2mFo - DFc map of mHv1cc (blue mesh) contoured at 1.0σ with the final model shows stick model. The map was calculated using native crystal (Cryst–B, S76–mHv1cc) at 3.45Å resolution. The magenta mesh shows the Bijvoet anomalous difference map of Zn²⁺ contoured at 5.0σ (20.00–5.0Å). The Zn²⁺ anomalous map was calculated from the native crystal data (Cryst–Z1) collected at 1.275Å wavelength using the phase as the refined coordinate of mHv1cc. (f) Stereo drawing of long helix (S4 and coiled–coil) in mHv1cc. The S4 consists of 3₁₀ helix (Arg201–Arg204) and α helix (Arg204–Arg207). Three arginine residues (Arg201, Arg204, and Arg207), and Met217 were drawn by stick model. The blue mesh shows the experimental map with MAD phases contoured at 1.0σ. The green mesh shows the Bijvoet anomalous difference map of selenomethionine (Met217) contoured at 5.0σ. The anomalous map of selenomethionine was calculated from MAD phases of Se-Met1 (Table 1). Since cell dimensions of Cryst–A were slightly deviated from those of Se-Met1, the refined model was fitted onto the Se–MAD map of Se-Met1 by rigid-body refinement. (g–i) Representative current traces in the presence of distinct doses of zinc ions. Data sets in each mutant [E115S(g), D119S(h), and ΔNΔC(i)] were recorded from the same patches. ΔNΔC denotes a version of mHv1 in which C-terminus and N-terminus were truncated at position 216 and 77, respectively¹⁶. Currents were elicited by test pulses to 100 mV under pH_out/pH_in = 7.0/7.0. The holding potential was -60 mV (black curve; 0 μM, red curve; 1 μM, and blue curve; 10 μM). (j, k) Sequence alignment of S4 (j) and comparison of S4 position among VSDs [mHv1cc (this structure, 3WKV)], Kv1.2 [2A79 (ref. 2)], KvAP (1ORS³⁹), and NavRh (4DXW³⁶) (k).