Extended Data Fig. 5: Views of the electron density map for PolF active site components in each of the six independent views provided in the asymmetric unit (ASU) of the Fe₂(II/II)•l-Ile•PolF complex.

(a) As in many other HDOs, PolF adopts a dimeric quaternary structure involving contacts between core α1 and aux α2. Interestingly, core α1 is interrupted by a long segment that adopts a loop-helix-loop structure. While other HDOs contain similar interruptions in this secondary structure, the PolF motif is the longest and most globular identified to date. In addition to composing the dimer, this segment also contacts core α3, potentially explaining why this component is ordered in all PolF crystals, even the mono-Zn(II) complex (Figure S23). (b) The ASU of Fe₂(II/II)•l-Ile•PolF contains six copies, arranged as three of the dimers shown in panel a, offering six independent views of the metal-binding site. Selected amino acids are shown as sticks, water molecules are shown as red spheres, and iron ions are shown as orange spheres. 2F_o-F_c maps are shown in gray mesh and contoured at 1.0σ. F_o-F_c maps are shown in red/green mesh and contoured at ±3.0σ. All chains show strong metal ion (71-100%) occupancy. In some chains, the electron density associated with Fe1 can be best modelled as glycerol (chains A and C), present in both the protein storage buffer and cryoprotectant solution (10-20% of the solution). Glycerol is similar in size to l-Ile and we assessed both options to account for the extra electron density at Fe1. Attempts to model l-Ile in chains A and C resulted in negative difference density associated with the carboxylic acid motif and poor density for the ethyl moiety of the side chain, two distinctive features of the substrate that can be used to differentiate from glycerol. The remaining chains show strong density for the carboxylate of l-Ile but varying coverage of the side chain. The side chain is most well-defined in chains D and F. Interestingly, in chain B, the side chain models best in a different rotameric form. This difference could reflect a capacity to accommodate different rotamers. It could also reflect a mixture of l-Ile and glycerol in this chain. Chain F was judged to be the highest quality model of the l-Ile substrate with the smallest contribution from glycerol. Consequently, it is used as the representative view in all other figures.