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  1. Evaluation Summary:

    This manuscript reveals new molecular details about the human calcium sensing receptor (CaSR), a G protein-coupled receptor that maintains calcium homeostasis and is involved in pathological states. This study, together with other recent structural work on this molecule, will have important implications for the design of molecules that control CaSR activity and treat human disease.

    (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):

    The data in this manuscript describe a previously unseen conformation of CaSR in complex with a nanobody that has been shown to inhibit calcium activation in functional experiments. Structural studies show that the nanobody physically prevents the dimerization of the extracellular domains. Although the structure of the nanobody-bound, putative inactive conformation is at relatively low resolution, the global conformational changes can be resolved. Such a nanobody may prove to be very useful as a antagonist of CaSR activation.

    Additional information and controls are required to support some of the main claims. The mutants are tested using an intracellular flux assay, but there are no controls to show that the mutant proteins have expressed and trafficked to the membrane. For mutants that inhibit function, cell surface expression controls are essential to establish that the effect of the mutant is specific.

    Additional details are required to evaluate the cryo-EM data, such as an FSC curve and the distribution of viewing angles of the particles used in the reconstruction. To evaluate the maps compared to the model, more extensive maps are required, especially in the regions where calcium ions and ligands are modelled. (Although the maps for the ligands are shown the surrounding regions are not). In Figure 1-Fgure Supplement 2, this appears to be a surface rendering rather than a map as indicated in the title of the figure. This rendering is more featureful than the provided maps, especially for the inactive conformation. In addition, there is no analysis of the quality of the model (bonds, angles, C-beta deviations, etc).

    This is presented as a more valid structure of the inactive state, compared to the "intermediate" state previously reported by Ling et al., 2021, Cell Res. 31: 383-394. This prior model suggested a conformational ensemble of inactivated states with varying degrees of separation of the extracellular domains. However, it is possible the nanobody in the present study simply stabilizes one state from this conformational ensemble and that there are other inactive states as well, such as those reported by Ling et al.

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  3. Reviewer #2 (Public Review):

    This manuscript reports the structure of the CaSR in both inactive and agonist+PAM bound states. Although a recent paper by Ling et al. (Cell Res 2021) first reported the structure of the CaSR in its inactive and active states, the present study adds novel and important information. The authors get good resolution of the CaSR extracellular domain (ECD), up to 3 A, allowing a better view of the Ca and amino acid binding sites. This paper is the first to report one of the important Ca site within the cleft of the VFT domain. Indeed, although several structures of the isolated ECD, and the structure of the full length CaSR were already reported, none really clarified the mode of action of Calcium ions leading to the activation of the receptor. However, the evidence that the density observed correspond to a Ca ion, rather than to another ion, remains to be confirmed.

    The second important information is the description of what is very likely the fully inactive state of the receptor, with both VFTs in the open state, and with a relative orientation leading to a large separation of the lobes 2 of the VFTs, as observed in the apo or antagonist bound states of most mGlu VFT dimers. Of special interest is the report of a nanobody that stabilize the inactive state of the CaSR, that was used to obtain the "inactive" structure. However, the possible influence of the nanobody in specifying this specific state is not being considered.

    The authors also identified a key element involved in the allosteric transition between the VFT and the 7TM domain, involving a contact between the C terminal part of the CRD and the ECL2 loop. This proposal based on the solved structures is validated by functional analysis of various CaSR mutants.

    A intriguing observation is that the 7TM conformation of both subunits within the dimer are almost identical in the "inactive" and ago+PAM state, indicating that the ago+PAM state is unlikely corresponding to the active conformation of the receptor. As such, the later state should not be named the "active" state, but rather the agonist+PAM state to be scientifically accurate.

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  4. Reviewer #3 (Public Review):

    In this study, the authors aim at deciphering the structural and molecular mechanisms of the human calcium sensing receptor function. For this purpose, they solved the cryoEM structures of inactive and active states of the receptor and provide further functional/pharmacological evidence to support the activation mechanism proposed from the structural data. Overall the data and approach are robust. It would be useful for the readers to better present/discuss the comparison of their structures with the recent structural data published recently for the same receptor (Ling et al 2021).

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