Structural dynamics of the human Orai1 channel revealed by cryo-electron microscopy

Yiming Zhang; Yuan Wang; Jindou Liu; Weiwei Bei; Hongkun Wang; Junli Wang; Lei Chen; Youjun Wang

doi:10.1371/journal.pone.0348440

Peer Review History

Original SubmissionJanuary 15, 2026
10 Feb 2026 Decision Letter - Jarrod B. French, Editor -->PONE-D-26-02314-->-->Structural dynamics of the human Orai1 channel revealed by cryo-electron microscopy-->-->PLOS One Dear Dr. Wang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.-->--> -->-->With respect to these revisions, we ask that you pay particular attention to structure/function questions posed by reviewer 2 and consider the suggested additional analyses to help clarify the findings of this work. In addition, please ensure that the content and quality of figures, and figure captions, is sufficient to clearly communicate your key findings (see reviewer 1 comments). Please submit your revised manuscript by Mar 27 2026 11:59PM. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. --> Reviewer #1: Yes Reviewer #2: Yes ******** -->2. Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: Yes Reviewer #2: I Don't Know ****** -->3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.--> Reviewer #1: Yes Reviewer #2: No ****** -->4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.--> Reviewer #1: Yes Reviewer #2: Yes ****** -->5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)--> Reviewer #1: This study reports the purification and initial cryo-EM characterization of the human Orai1 (hOrai1) channel, confirming its hexameric assembly and identifying substantial structural heterogeneity—with particles adopting both C6- and C2-symmetric conformations. These findings represent a valuable foundational step for structural investigations of this medically relevant ion channel. That said, the work remains in a preliminary state: the obtained resolution is moderate, the observed structural states are not functionally annotated, and the analysis lacks in-depth interpretation of the conformational dynamics within physiological or disease-relevant contexts. Several critical revisions are required to strengthen the manuscript for potential publication, as outlined below: 1. The core finding of this study is the coexistence of C6- and C2-symmetric hOrai1 particles, indicative of intrinsic structural dynamics. Notably, this very heterogeneity is attributed to the limited resolution (~4.5–5.0 Å), which precludes atomic model building. A major knowledge gap is the complete absence of functional data to assign biological relevance to these distinct conformational states. Key unanswered questions include: Is the C2-symmetric state a gating intermediate en route to channel opening, an inactivated state, or a detergent-induced artifact of sample preparation? The discussion section must be expanded to address these critical questions and contextualize this core finding. 2. Figure quality and accompanying legends require significant improvement. For Fig. 4 (the most analysis-intensive panel, featuring comparisons with dOrai states), the legend is insufficiently descriptive and must explicitly define the meaning of red arrows (e.g., “red arrows indicate the direction of M1 helix movement associated with channel opening”). Additionally, it is recommended that Fig. S1 be moved to the main text as a new Fig. 2 to elevate the presentation of key supporting data. 3. A typographical error is present on Page 4, Line 3: the phrase “We next expressed hOrai1-ΔN-strep and and solubilized it in LMNG-CHS mixed micelles” contains a redundant “and”. This should be corrected to: “We next expressed hOrai1-ΔN-Strep and solubilized it in LMNG-CHS mixed micelles”. 4. A duplication exists in the reference list: Reference #10 and Reference #17 are identical, both citing Lu, X. et al. (2024) Biochem. Biophys. Res. Commun. 733, 150723. This redundancy must be removed, and the reference numbering adjusted accordingly. Reviewer #2: The authors have resolved for the first time the hOrai1 structure however with weak resolution and not much detailed structural insight. They reveal two resolutions maps C2 and C6, while C6 is the fully symmetric one. Further they perform comparisons with the closed and open dOrai structures and suggest that their hOai1 structure matches more with the closed dOrai structure. While the resolution of hOrai1 structure is of course of big interest in the community, many open questions in terms of structural details remain. Major: Is the C2 map a potential physiological state? They authors also mention that they found pentameric populations. Can you further elaborate on the physiological relevance of the different states/maps found?- Please further comment on the resolution and structure of the pore. Can the authors comment on the CT in the hOrai1 structures. What is the predicted orientation? Similarly for TM4 – Can the authors predict whether there is kink in TM4? The authors compare their structure with close dOrai (6BBG) showing fully straightend CT. Why did they chose this structure and not one with the antiparallel crossing CTs? Please also perform a comparison with 4HKR. ****** -->6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.--> Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. --> https://doi.org/10.1371/journal.pone.0348440.r001
Revision 1
12 Apr 2026 Author Response Response to Reviewers’ comments Please note that our responses to the reviewers’ comments were shown in blue below. Our changes in the manuscript text were shown in red. Reviewer #1: This study reports the purification and initial cryo-EM characterization of the human Orai1 (hOrai1) channel, confirming its hexameric assembly and identifying substantial structural heterogeneity—with particles adopting both C6- and C2-symmetric conformations. These findings represent a valuable foundational step for structural investigations of this medically relevant ion channel. That said, the work remains in a preliminary state: the obtained resolution is moderate, the observed structural states are not functionally annotated, and the analysis lacks in-depth interpretation of the conformational dynamics within physiological or disease-relevant contexts. Several critical revisions are required to strengthen the manuscript for potential publication, as outlined below: Specific comments: 1. The core finding of this study is the coexistence of C6- and C2-symmetric hOrai1 particles, indicative of intrinsic structural dynamics. Notably, this very heterogeneity is attributed to the limited resolution (~4.5–5.0 Å), which precludes atomic model building. A major knowledge gap is the complete absence of functional data to assign biological relevance to these distinct conformational states. Key unanswered questions include: Is the C2-symmetric state a gating intermediate en route to channel opening, an inactivated state, or a detergent-induced artifact of sample preparation? The discussion section must be expanded to address these critical questions and contextualize this core finding. Response: We thank the reviewer for this insightful comment. To be clear, we think the structural heterogeneity is the reason for the limited resolution. We provided the Supplementary Movie 1 showing the continuous structural heterogeneity in the sample in the revised manuscript. We agree that, at the current resolution (~4.5–5.0 Å), our data do not allow definitive assignment of the observed C6- and C2-symmetric hOrai1 conformations to specific functional states. In the revised Discussion, we have added the following sentences: “Notably, we observed a continuous distribution between C2 and C6 conformations, indicating that these states interconvert rather than existing as discrete populations. This continuous structural heterogeneity posed a major technical obstacle to achieving high-resolution reconstruction and hindered accurate model building. At the current resolution (~4.5–5.0 Å), the maps do not permit definitive model building or functional assignment of the observed conformational states.” we have clarified this limitation and avoided overinterpretation of the biological relevance of these structures. We also added the following content to the Discussion section: “Importantly, the C2-symmetric particles retain a six-subunit architecture, ruling out partial subunit dissociation or gross misassembly. The presence of intermediate conformations connecting C2 and C6 populations during 2D and 3D classification further supports the idea that these states are interconverting. However, it remains unclear whether the coexistence of C2 and C6 populations reflects a thermodynamic equilibrium of hOrai1, a transient gating-related or inactivated conformation, or a state stabilized by the detergent environment. Although contributions from the detergent cannot be fully excluded, the observation of continuous transitions between C2 and C6 argues against a purely sporadic or damage-related origin. Our results suggest that stabilizing the C6 population through sample optimization or construct modification will be essential for improving map resolution and enabling detailed structural and functional analysis.”. We further explicitly state that the C2-symmetric particles cannot be conclusively classified as “a transient gating-related or inactivated conformation, or a state stabilized by the detergent environment”. Consistent with the scope of PLOS ONE, we present these observations as structural evidence of conformational variability without assigning a specific physiological role. We have revised the Discussion accordingly to clearly delineate the limits of interpretation and to outline future approaches required to establish functional relevance. 2-1. Figure quality and accompanying legends require significant improvement. Response: We thank the reviewer for this helpful suggestion. To enhance clarity and professionalism, we have reprocessed all images at high resolution, standardized the font sizes, and improved the color contrast for better visibility. Furthermore, we have substantially revised all figure legends to ensure they are clear. 2-2. For Fig. 4 (the most analysis-intensive panel, featuring comparisons with dOrai states), the legend is insufficiently descriptive and must explicitly define the meaning of red arrows (e.g., “red arrows indicate the direction of M1 helix movement associated with channel opening”). Response: We thank the reviewer for the reminder. We have specified in the figure legend that “The red arrows indicate the relative movement trend of the TM1-3 helices associated with channel opening.” 2-3. Additionally, it is recommended that Fig. S1 be moved to the main text as a new Fig. 2 to elevate the presentation of key supporting data. Response: We thank the reviewer for the suggestion. We have moved some panels in Fig. S1 to Fig. 3 and revised the numbering of all figures in the manuscript. 3. A typographical error is present on Page 4, Line 3: the phrase “We next expressed hOrai1-ΔN-strep and and solubilized it in LMNG-CHS mixed micelles” contains a redundant “and”. This should be corrected to: “We next expressed hOrai1-ΔN-Strep and solubilized it in LMNG-CHS mixed micelles”. Response: We thank the reviewer for the correction. The text has been updated as follows: “We next expressed hOrai1-ΔN-strep and solubilized it in LMNG-CHS mixed micelles. The protein was purified via Streptactin affinity chromatography and size-exclusion chromatography (SEC), which showed a monodisperse peak (Fig. 1B). ” 4. A duplication exists in the reference list: Reference #10 and Reference #17 are identical, both citing Lu, X. et al. (2024) Biochem. Biophys. Res. Commun. 733, 150723. This redundancy must be removed, and the reference numbering adjusted accordingly. Response: We thank the reviewer for noting this, and the error has been corrected. Reviewer #2: The authors have resolved for the first time the hOrai1 structure however with weak resolution and not much detailed structural insight. They reveal two resolutions maps C2 and C6, while C6 is the fully symmetric one. Further they perform comparisons with the closed and open dOrai structures and suggest that their hOai1 structure matches more with the closed dOrai structure. While the resolution of hOrai1 structure is of course of big interest in the community, many open questions in terms of structural details remain. Major: 1. Is the C2 map a potential physiological state? They authors also mention that they found pentameric populations. Can you further elaborate on the physiological relevance of the different states/maps found? Response: We thank the reviewer for this important question. At the current resolution, we cannot definitively assign the C2-symmetric map to a specific physiological state. In the revised Discussion, we clarify that the C2 population may represent one of several possibilities, including a thermodynamically accessible conformational state, a transient gating- or inactivation-related conformation, or a state stabilized under detergent conditions. We emphasize that no definitive functional assignment is made. And added the following statement in the Discussion: "At the current resolution (~4.5–5.0 Å), the maps do not permit definitive model building or functional assignment of the observed conformational states." Importantly, the C2-symmetric particles retain a complete hexameric architecture and appear in a continuum with the C6 population during 2D and 3D classification, suggesting reproducible conformational variability rather than sporadic misassembly. We have added the following discussion in the corresponding section: "Importantly, the C2-symmetric particles retain a six-subunit architecture, ruling out partial subunit dissociation or gross misassembly. The presence of intermediate conformations connecting C2 and C6 populations during 2D and 3D classification further supports the idea that these states are interconverting." Regarding the pentameric population of hOrai1, we note that pentameric assemblies of hOrai1 have previously been reported in hOrai1–SOAR fusion constructs. We have added the following statement in the Discussion section: "In our dataset, however, the pentamer represents only a minor population, while the hexamer is predominant, suggesting that under the present experimental conditions, the hexamer is the dominant assembly state of hOrai1." We speculated the pentameric species are assembly intermediate of hOrai hexamer. 2. Please further comment on the resolution and structure of the pore. Response: The moderate resolution in the current maps limits detailed structural interpretation of the pore. In the revised manuscript, we clarify that the density corresponding to the pore-lining M1 helices is clearly identifiable and allows assignment of the overall pore architecture, but the map quality does not support reliable side-chain positioning or precise measurement of pore radius. We therefore refrain from detailed mechanistic interpretation of ion permeation. The text has been revised to “pore-lining helices” to better reflect these limitations. 3. Can the authors comment on the CT in the hOrai1 structures. What is the predicted orientation? Similarly for TM4 – Can the authors predict whether there is kink in TM4? Response: We have supplemented additional Supplementary Fig. 1C-D to show the densities of the M4 helices and CT. As a reference, we also provided the dOrai structure in the revised Supplementary Fig. 1E. At the current resolution, the density corresponding to the C-terminal region is relatively weak and partially discontinuous, precluding definitive modeling of its structures. 4. The authors compare their structure with close dOrai (6BBG) showing fully straightend CT. Why did they chose this structure and not one with the antiparallel crossing CTs? Please also perform a comparison with 4HKR. Response: The M4 helix in 4HKR adopts a latched conformation not observed in any other dOrai structures, including 6BGG, 6BBF, 6BBH, 6BBI, 6AKI, 7KR5. Examination of the crystal packing in 4HKR reveals that the latched M4 is heavily involved in crystal contacts (Fig. 1). Thus, we speculate that the latched M4 conformation in 4HKR may result from crystal packing and may not be suitable for structural comparison with hOrai1. Fig. 1 Crystal structure of dOrai (PDB: 4HKR). Three dOrai hexamers are shown in gray, with their TM4 helices involved in crystal packing highlighted in yellow, purple, and green, respectively. Attachments Attachment Submitted filename: Response to Reviewers-hOrai-03-05-zhang et al.docx https://doi.org/10.1371/journal.pone.0348440.r002
16 Apr 2026 Decision Letter - Jarrod B. French, Editor Structural dynamics of the human Orai1 channel revealed by cryo-electron microscopy PONE-D-26-02314R1 Dear Dr. Wang, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jarrod B. French, PhD Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0348440.r003
Formally Accepted
Acceptance Letter - Jarrod B. French, Editor PONE-D-26-02314R1 PLOS One Dear Dr. Wang, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Jarrod B. French Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0348440.r004

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