Extended Data Fig. 4: X-ray crystal structure of Dy^III-bound Hans-LanM, solved at 1.4 Å resolution.

a, One of the dimers in the asymmetric unit, comprising chains A and B. Note that EF4 is unexpectedly occupied with Dy^III while EF1 is occupied only in chain A. b, Overall structure of the asymmetric unit, which consists of two Hans-LanM dimers. Unlike the La^III-Hans-LanM structure, the two dimers – and the monomers within each dimer – display significant differences in Dy^III-Hans-LanM. EF2-4 are occupied by Dy^III in all chains, whereas EF1 is only occupied and ordered in chain A; in chains B and C, no metal ion is bound in the EF-hand, and in chain D, a Dy^III ion is bound but the first five residues of EF1 (Asn34 – Asp38) could not be modeled. Our decision to model Dy^III into all four EF-hands is supported by anomalous diffraction datasets (Supplementary Tables 9–10, Supplementary Figs. 25–26). The biochemical data suggest that, in solution, at least one Dy^III binding site is weak (see Fig. 1d and Supplementary Fig. 2), and it is likely based on studies of Mex-LanM that EF2/3 are the tighter binding sites. This proposal is supported by the Dy anomalous data (Supplementary Table 10), and the occupancy of weak metal-binding sites likely results from the high protein concentration used for crystallography. c, Details of metal coordination in the EF-hands of Dy^III-Hans-LanM. In the top row, the three different EF1 structures in the asymmetric unit are shown. Only in chain A is the EF1 metal site nearly identical to the sites in EF2 and EF3 (contrary to La^III-Hans-LanM, where EF1-3 sites are very similar, Extended Data Fig. 3). In EF1 (chain A), EF2, and EF3, the coordinating ligands are the same as with La^III-Hans-LanM, except that the E₉ residues (Glu42, Glu66, and Glu91) have shifted to monodentate coordination, resulting in 9-coordination. The lower coordination number with Dy^III is consistent with the lanthanide contraction^68,69 and is observed with other ligands (as one recent example, ref. ³⁹). The Dy^III-ligand distances are mostly 2.3-2.5 Å, ~0.2 Å shorter than for La^III-Hans-LanM. Consistent with this observation, the crystal radius for 9-coordinate Dy^III is given as 1.22 Å by Shannon⁷, 0.19 Å shorter than for 10-coordinate La^III (Extended Data Fig. 3). The carboxylate shift of the 9^th position Glu residue is noteworthy as this position is important for gating affinity and selectivity in other EF-hand proteins⁷⁰. In EF4, Dy^III is 7-coordinate with pentagonal bipyramidal geometry, similar to the sodium site in La^III-Hans, but with slightly shorter metal-ligand distances (2.2-2.5 Å); again, these distances are consistent with the expectation for 7-coordinate Dy^III (ref. ⁷).