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  1. Evaluation Summary:
    This work elegantly fuses cryo-EM, x-ray crystallography, and in vitro transport experiments to describe the structural basis for functional diversity in the SLC11/NRAMP family of membrane transporters. This work identifies factors responsible for selectivity of classical NRAMPS for transition metal ions (Fe, Mn) and the NRMT clade for alkali metal ion (Mg). Although selectivity is much discussed in transport of divalent metal ions, this is an outstanding example of a study that gets to the bottom of the structural determinants governing this behavior.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    This manuscript describes the structural and functional analysis of a discrete clade of magnesium transporters related to the well-studied NRAMP divalent metal ion transporters. The structural approach - cryo-EM for high resolution structure determination coupled with crystallography to precisely identify ion binding sites via anomalous scattering - is excellent, combining the different strengths of the two techniques. The structures clearly represent the culmination of a lot of tenacious biochemical work to achieve a structurally tractable, nanobody-stabilized transporter.

    The structures are coupled with thoughtful and extensive substrate transport data to investigate the effects of various mutants on substrate transport. The authors make the surprising observation that Mg2+ transport is not coupled to proton movement. However, they make a convincing case that, in the physiological context, the electrical and chemical gradients are such that proton-coupling is not energetically required, and structural observations corroborate this interpretation. Although the functional experiments were mostly thorough and well-controlled, it appears that different membrane potentials were applied in proton- and metal-uptake experiments, and the functional insights could be augmented by considering how these potentials might influence transport and controlling for the membrane potential.

    Together, these data complement existing functional and structural observations for other members of the SLC11 family with different substrate specificities, leading to a comprehensive mechanistic picture of divalent metal ion binding and selectivity among this family of transporters.

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  3. Reviewer #2 (Public Review):

    There are two important findings in this manuscript. First, the authors discussed the unique ion selectivity property of prokaryotic NRAMPs for Mg2+ and proposed that Mg2+ would be recognized in a nearly fully-hydrated form. Second, based on the proteoliposome experiments, the authors show that EleNRMT is not driven by proton and maybe more like a uniporter. Based on the structural comparison, they proposed the replacement of His with Trp may be crucial for this difference. I basically agree with these views and have several comments as shown below.

    1. I basically agree that EleNRMT would recognize Mg2+ ion in a nearly fully-hydrated form, but to conclude it, the resolution of the EleNRMT structures is a kind of moderate. I think extra efforts, such as the MD simulation, would be too much work and not necessary, but it would be helpful if the authors can mention the limitation of the resolution of the present structures, where water molecules are not clearly visible, somewhere in the manuscript.

    2. As this is a great success of the thermostabilization of membrane proteins, it would be helpful to other people in this research field if the authors provide a more detailed protocol for the thermal shift assays (Figure 3C).

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  4. Reviewer #3 (Public Review):

    The paper begins with a robust set of transport assays comparing the ion specificity of an E. coli NRAMP (EcoDMT) with an NRMT from the bacterium Eggerthella lenta (EleNRMT). These assays confirm Mn transport for both proteins, though EleNRMT has 10-fold lower affinity and a notable competition with Mg. Mutation of active site residues previously proposed to account for these differences fail to convert EcoDMT into a Mg transporter, although these mutations do allow Ca transport. This result provides a strong premise for structural studies of NRMT.

    EleNRMT was chosen from a broad screen of NRMT homologs and was then optimized for structural studies by the introduction of 3 thermostabilizing mutations. The authors have been careful to document that thermostabilization has not significantly altered Mn transport by the protein. They document the expected Mg transport activity together with a lack of Ca transport. Thus, despite having the same 4 active site residues as the mutated EcoDMT, EleNRMT has distinct transport properties which call for detailed structural studies of the ion binding site.

    To facilitate cryo-EM on such a small target (~50kD), nanobodies were raised by immunization of alpacas followed by phage-display selection. They settled on imaging a remarkable trimeric complex composed of NRMT and two of the nanobody binders, which led not only to two cryo-EM structures in the presence and absence of Mg, but also to anomalous X-ray scattering data from modestly ordered crystals. The cryo-EM structures define the architecture of the protein, though the modest resolution of the Mg-bound structure seems to limit definition of the site. Nevertheless, anomalous X-ray scattering definitively confirms the location of the ion binding site. The molecule adopts an inward-facing conformation of NRMT that is very similar to previous work on NRAMPs. However upon closer inspection, the authors describe an expanded ion binding site that is able to accommodate a hydrated Mg ion with a coordination shell that clearly differs from the dehydrated Mn ion seen in NRAMPs. Although it is not explicitly discussed, the transport data imply that the binding site of EleNRMT is flexible enough to adopt either configuration depending of the ligand it encounters.

    Armed with this structure, the authors introduce mutations to several key residues within the ion binding pocket. The results from transport assays confirm the authors' ideas about Mg selectivity and ultimately bring the paper full circle by generating the Ca permeability as seen in the mutated EcoDMT protein.

    Although the functional data clearly show the lack of proton coupling in EleNRMT, the structural explanation is not so convincing. It is not clear that the proposed proton pathway for NRAMPS is so specific that the authors conclusion about the replacement of His228 with a Trp is clear cut. This issue seems complex and deserves further discussion and perhaps mutational data to support the critical importance of this His residue.

    However, taken together, the extensive set of results presented in this paper provide a robust and comprehensive explanation of ion selectivity amongst the broader SCL11/NRAMP family.

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