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Architecture of the human KICSTOR and GATOR1–KICSTOR complexes

Nature Structural & Molecular Biology volume 32, pages 2587–2600 (2025)Cite this article

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Abstract

The human KICSTOR complex, comprising KPTN, ITFG2, C12orf66 and the scaffolding protein SZT2, anchors the mTORC1 inhibitor GATOR1 to lysosomes. Mutations affecting KICSTOR subunits are associated with severe neurodevelopmental and epileptic disorders. Loss of KICSTOR mimics GATOR1 inactivation, resulting in constitutive mTORC1 activation, highlighting its critical role in nutrient sensing. Here, we used cryo-electron microscopy and computational modeling to determine the architectures of KICSTOR and the GATOR1–KICSTOR supercomplex. We show that SZT2 forms a crescent-shaped scaffold with repetitive tandem units, binding the ITFG2–KPTN heterodimer and C12orf66 at its C terminus. Structural and biochemical analyses revealed that GATOR1 binds the SZT2 N-terminal domain through NPRL3; disruption of this interaction hyperactivates mTORC1 and mislocalizes TFE3 independently of nutrient status. We further demonstrate the membrane-binding ability of KICSTOR, with SZT2 and C12orf66 preferentially interacting with negatively charged lipids—a requirement for lysosomal localization. These findings identify how KICSTOR positions GATOR1 on lysosomes to regulate nutrient-dependent mTORC1 signaling.

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Fig. 1: Cryo-EM structure of the human KICSTOR complex.
Fig. 2: Structure of the KICSTOR subunits.
Fig. 3: Cryo-EM structure of the human GATOR1–KICSTOR complex.
Fig. 4: Interaction of human GATOR1–KICSTOR complex and influence in mTORC1 signaling.
Fig. 5: KICSTOR does not impact the GAP activity of GATOR1 toward Rag GTPases.
Fig. 6: The membrane interactions of the KICSTOR complex.
Fig. 7: Characterization of KICSTOR lysosomal targeting.

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Data availability

The cryo-EM density maps were deposited to the Electron Microscopy Data Bank under accession codes EMD-64799 (KICSTOR in state 1), EMD-64902 (KICSTOR in state 2), EMD-64831 (KICSTOR CCC in state 3), EMD-64827 (KICSTOR CCC in state 4), EMD-64877 (KICSTOR CCC in state 5) and EMD-64665 (KICSTOR–GATOR1 complex), with corresponding atomic coordinates available in the PDB under accession numbers 9V6E (KICSTOR in state 1), 9VAN (KICSTOR in state 2), 9V86 (KICSTOR CCC in state 3), 9V80 (KICSTOR CCC in state 4), 9V9N (KICSTOR CCC in state 5) and 9V0J (KICSTOR–GATOR1), respectively. MS proteomics data are accessible from ProteomeXchange (PXD060391) through the PRIDE repository. Source data are provided with this paper.

References

  1. Lim, C. Y. & Zoncu, R. The lysosome as a command-and-control center for cellular metabolism. J. Cell Biol. 214, 653–664 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 169, 361–371 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Schurmann, A., Brauers, A., Massmann, S., Becker, W. & Joost, H. G. Cloning of a novel family of mammalian GTP-binding proteins (RagA, RagBs, RagB1) with remote similarity to the RAS-related GTPases. J. Biol. Chem. 270, 28982–28988 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Sekiguchi, T., Hirose, E., Nakashima, N., Ii, M. & Nishimoto, T. Novel G proteins, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B. J. Biol. Chem. 276, 7246–7257 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Inoki, K., Li, Y., Xu, T. & Guan, K. L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Gene Dev. 17, 1829–1834 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sancak, Y. et al. Ragulator–Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Anandapadamanaban, M., et al. Architecture of human Rag GTPase heterodimers and their complex with mTORC1. Science 366, 203–210 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rogala, K. B. et al. Structural basis for the docking of mTORC1 on the lysosomal surface. Science 366, 468–475 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Panchaud, N., Peli-Gulli, M. P. & De Virgilio, C. Amino acid deprivation inhibits TORC1 through a GTPase-activating protein complex for the Rag family GTPase Gtr1. Sci. Signal. 6, ra42 (2013).

    Article  PubMed  Google Scholar 

  12. Valenstein, M. L. et al. Structure of the nutrient-sensing hub GATOR2. Nature 607, 610–616 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Su, M. Y., et al. Cryo-EM structures of amino acid sensors bound to the human GATOR2 complex. Cell Rep. 44, 116088 (2025).

    Article  CAS  PubMed  Google Scholar 

  14. Peng, M., Yin, N. & Li, M. O. SZT2 dictates GATOR control of mTORC1 signalling. Nature 543, 433–437 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wolfson, R. L. et al. KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. Nature 543, 438–442 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Baple, E. L. et al. Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures. Am. J. Hum. Genet 94, 87–94 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Holm, L., Laiho, A., Toronen, P. & Salgado, M. DALI shines a light on remote homologs: one hundred discoveries. Protein Sci. 32, e4519 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu, H. et al. Crystal structure of human spermine synthase: implications of substrate binding and catalytic mechanism. J. Biol. Chem. 283, 16135–16146 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sette, P. et al. The Phe105 loop of Alix Bro1 domain plays a key role in HIV-1 release. Structure 19, 1485–1495 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ramamurthy, V. et al. Structures of adnectin/protein complexes reveal an expanded binding footprint. Structure 20, 259–269 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Su, M. Y. et al. Hybrid structure of the RagA/C–Ragulator mTORC1 activation complex. Mol. Cell 68, 835–846 e833 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Egri, S. B. et al. Cryo-EM structures of the human GATOR1–Rag–Ragulator complex reveal a spatial-constraint regulated GAP mechanism. Mol. Cell 82, 1836–1849 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Shen, K. et al. Architecture of the human GATOR1 and GATOR1–Rag GTPases complexes. Nature 556, 64–69 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Qu, A. & Leahy, D. J. Crystal structure of the I-domain from the CD11a/CD18 (LFA-1, αLβ2) integrin. Proc. Natl Acad. Sci. USA 92, 10277–10281 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Liu, M. et al. Structure of the DDB1–AMBRA1 E3 ligase receptor complex linked to cell cycle regulation. Nat. Commun. 14, 7631 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Merkley, E. D. et al. Distance restraints from crosslinking mass spectrometry: mining a molecular dynamics simulation database to evaluate lysine–lysine distances. Protein Sci. 23, 747–759 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tafur, L. et al. Cryo-EM structure of the SEA complex. Nature 611, 399–404 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wolfson, R. L. & Sabatini, D. M. The dawn of the age of amino acid sensors for the mTORC1 pathway. Cell Metab. 26, 301–309 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kim, W., Underwood, R. S., Greenwald, I. & Shaye, D. D. OrthoList 2: a new comparative genomic analysis of human and Caenorhabditis elegans genes. Genetics 210, 445–461 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cattelani, C. et al. The SZT2 interactome unravels new functions of the KICSTOR complex. Cells 10, 2711 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tan, S. M. The leucocyte β2 (CD18) integrins: the structure, functional regulation and signalling properties. Biosci. Rep. 32, 241–269 (2012).

    Article  CAS  PubMed  Google Scholar 

  33. Sbrissa, D., Ikonomov, O. C., Filios, C., Delvecchio, K. & Shisheva, A. Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636. Am. J. Physiol. Cell Physiol. 303, C436–C446 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nakatsu, F. & Kawasaki, A. Functions of oxysterol-binding proteins at membrane contact sites and their control by phosphoinositide metabolism. Front. Cell Dev. Biol. 9, 664788 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Wallroth, A. & Haucke, V. Phosphoinositide conversion in endocytosis and the endolysosomal system. J. Biol. Chem. 293, 1526–1535 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Hasegawa, J., Strunk, B. S. & Weisman, L. S. PI5P and PI(3,5)P: minor, but essential phosphoinositides. Cell Struct. Funct. 42, 49–60 (2017).

    Article  CAS  PubMed  Google Scholar 

  37. Fitzian, K., et al. TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation. Mol. Cell 81, 2705–2721 (2021).

    Article  CAS  PubMed  Google Scholar 

  38. Ebner, M., et al. Nutrient-regulated control of lysosome function by signaling lipid conversion. Cell 186, 5328–5346 (2023).

    Article  CAS  PubMed  Google Scholar 

  39. Hong, Z. et al. PtdIns3P controls mTORC1 signaling through lysosomal positioning. J. Cell Biol. 216, 4217–4233 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  42. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Cianfrocco, M. A. COSMIC: a global resource for structure prediction and structural analysis. Biophys. J. 123, 334a–335a (2024).

    Article  Google Scholar 

  45. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  46. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  49. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  51. Su, M. Y., Fromm, S. A., Remis, J., Toso, D. B. & Hurley, J. H. Structural basis for the ARF GAP activity and specificity of the C9orf72 complex. Nat. Commun. 12, 3786 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Bolte, S. & Cordelières, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Perez-Riverol, Y. et al. The PRIDE database at 20 years: 2025 update. Nucleic Acids Res. 53, D543–D553 (2024).

    Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to H. Rao and F. Rao for their valuable comments on the manuscript. M.-Y.S. is an investigator of SUSTech Institute for Biological Electron Microscopy. We thank the following facilities for their technical support: the Cryo-EM Center at SUSTech, the Cryo-EM Facility and the Advanced MS Facility of the Kobilka Institute of Innovative Drug Discovery at the Chinese University of Hong Kong (Shenzhen), Shenzhen Medical Academy of Research and Translation (SMART) for the assistance in MST data collection. This work was supported by funding from the Shenzhen Medical Research Fund (B2402014 to G.S. and B2502011 to M.-Y.S.), National Natural Science Foundation of China (32571412 to M.-Y.S., 32170779 to C.-Y.L.), the Natural Science Foundation of Guangdong Province (2024A1515011683 to M.-Y.S.), Shenzhen Science and Technology Program (20231120103446003 to M.-Y.S.), National High-level Talents Program (HJJH22-003 to C.-Y.L.), CUHK-Shenzhen University Development Fund (to G.S.), Medical Research Innovation Project G030410001 and start-up funding from SUSTech (to M.-Y.S.).

Author information

Author notes

  1. These authors contributed equally: Fei Teng, Huan Zeng.

Authors and Affiliations

  1. Department of Biochemistry, Key University Laboratory of Metabolism and Health of Guangdong, SUSTech Homeostatic Medicine Institute, School of Medicine, Southern University of Science and Technology, Shenzhen, China

    Fei Teng, Huan Zeng, Shuyun Tian, Shan Wang & Ming-Yuan Su

  2. Kobilka Institute of Innovative Drug Discovery, School of Medicine, The Chinese University of Hong Kong, Shenzhen, Shenzhen, China

    Xinyi Mai & Goran Stjepanovic

  3. School of Basic Medical Sciences, Guangzhou National Laboratory, Guangzhou Medical University, Guangzhou, China

    Shujun Chen, Lulu Wang, Zeming Feng & Chun-Yan Lim

  4. MOE Key Laboratory of Gene Function and Regulation, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Lulu Wang

  5. College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

    Zeming Feng

  6. Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, China

    Ming-Yuan Su

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Contributions

M.-Y.S. conceptualized and supervised the project, designed the experimental strategy and performed the biochemical and structural studies. F.T. and H.Z. conducted the biochemical assays and cellular experiments. X.M. performed the cryo-EM sample screening, data collection and liposome-binding assays. S.C., L.W. and Z.F. executed the cell biological experiments. S.T. and S.W. provided technical assistance. G.S., C.-Y.L. and M.-Y.S. designed the study, interpreted the results and wrote the manuscript with input from all authors.

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Correspondence to Goran Stjepanovic, Chun-Yan Lim or Ming-Yuan Su.

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Nature Structural & Molecular Biology thanks Volker Haucke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Cryo-EM structure determination of the full-length KICSTOR complex.

a, Representative motion-corrected cryo-EM micrograph (of 30,943 micrographs from 4 datasets) of the full-length human KICSTOR complex. b, Representative 2D class averages of the full-length KICSTOR complex. c, Workflow for cryo-EM data processing of full-length KICSTOR datasets. NU-refinement: non-uniform refinement. d, FSC plots for the consensus reconstruction of the full-length KICSTOR in state 1 are between two independently refined half-maps with no mask (blue), spherical mask (orange), loose mask (green), and tight mask (red). A cutoff value of 0.143 was used to estimate the resolution. e, Angular particle distribution for projection views of full-length KICSTOR in state 1, calculated using CryoSPARC. The heat map shows the number of particles for each viewing angle. f, Full-length KICSTOR in state 1 density map, color-coded according to local resolution estimation. g-i, Results of NU-refinement for the consensus reconstruction of full-length KICSTOR in state 2. (g) FSC curves, (h) angular distribution heatmap, (i) local resolution estimation, with the map color-coded to reflect the resolution. j, Masking and local refinement applied to the SZT2SZ1-SZ2-SZ3 and SZT2SZ7-C12orf66 regions of full-length KICSTOR in state 1. k, Masking and local refinement applied to the SZT2SZ1-SZ2-SZ3 or SZT2SZ7-C12orf66 regions of full-length KICSTOR in state 2.

Extended Data Fig. 2 Overview of cryo-EM processing of the KICSTOR C-terminal core complex (CCC).

a, Superose 6 size-exclusion chromatogram profile of the human KICSTOR CCC. The peak corresponding to KICSTOR CCC is marked with a black asterisk. Coomassie blue-stained SDS-PAGE analysis of the purified KICSTOR. MW, molecular weight. mAU, milliabsorbance. Data in a is representative of at least three independent experiments. b, Representative motion-corrected cryo-EM micrograph (of 8,829 micrographs from 1 dataset) of the KICSTOR C-terminal core complex. c, Representative 2D class averages of the KICSTOR CCC. d, Workflow for cryo-EM data processing of the KICSTOR CCC. NU-refinement: non-uniform refinement. e-g. Half-map FSC plots, angular particle distributions and local resolution estimation from consensus reconstructions of the KICSTOR CCC for state 3 (class 1), state 4 (class 2), and state 5 (class 3). h-i. Masking and local refinement applied to the SZT2SZ7-C12orf66 regions of the KICSTOR CCC for state 3 and state 5. j, Individual composite maps generated for different states of the KICSTOR CCC, prepared for subsequent model building.

Extended Data Fig. 3 Structural comparison of full-length KICSTOR and KICSTOR CCC complex.

a-b, Fitting of the fulllength KICSTOR models into cryo-EM reconstructions for state 1 and state 2. Zoomed-in views of the SZT2SZ7-C12orf66 region are shown on the right. c-e, Fitting of the KICSTOR CCC models into cryo-EM reconstructions for state 3, state 4 and state 5. Corresponding zoomed-in views of the SZT2SZ7-C12orf66 portions are displayed on the right. f, Superimposition of the structural models from all states to illustrate conformational differences on SZT2SZ7-C12orf66. Dotted boxes indicate regions that are magnified in panels g-i. RMSD values for the superimposed structures are shown in Supplementary Table 2. g, Structural comparison of the SZT2SZ7-C12orf66 region between state 1 and state 2. The arrow indicates the rotation observed in the subunits. h, Structural comparison of the SZT2SZ7-C12orf66 region between state 1 and state 3. i, Structural comparison of the SZT2SZ7-C12orf66 region between state 1 and state 5.

Extended Data Fig. 4 Model overlays of SZT2 and C12orf66.

a, Overlay of SZT2SZ1 (light gray) with spermine synthase (light sky blue, PDB 3C6K), SZT2NTD, Depdc5NTD-SABA domains (forest green, PDB 7T3B), and CD11a I domain (rosy brown, PDB 1LFA). RMSD values for the superimposed structures are shown in Supplementary Table 2. b, Overlay of C12orf66NTD (orange) with BroxBro1 (cornflower blue, PDB 3R9M), SRP68 (pink, PDB 7QWQ), C12orf66roadblock (orange), and Lamtor2 (purple, PDB 6B9X). RMSD values for the superimposed structures are shown in Supplementary Table 2. c, Close-up view of the SZT2-C12orf66 interface. W3428 of SZT2 is required to interact with C12orf66 L216 and F269, as shown by the pulldown assay in d-e. d, Pulldown experiment of wild-type or mutated SZT2 with KPTN, ITFG2, and C12orf66. TSF, twin-strep-flag. e, Pulldown experiment of wild-type or mutated C12orf66 with KPTN, ITFG2, and SZT2. TSF, twin-strep-flag. Data in d and e are representative of three independent experiments.

Source data

Extended Data Fig. 5 Cryo-EM structure determination of the human GATOR1-KICSTOR supercomplex.

a, EM image of the negatively stained GATOR1-KICSTOR supercomplex. The shape of the supercomplex can be recognized and circled in the raw image. Data in a is representative of at least three independent experiments. b, Representative motion-corrected cryo-EM micrograph (of 9,936 micrographs from 1 dataset) of the human GATOR1-KICSTOR complex. c, Representative 2D class averages for the GATOR1-KICSTOR supercomplex. d, Workflow for the cryo-EM data processing of human GATOR1-KICSTOR complex. NU-refinement: non-uniform refinement. e-g, FSC plots (e), angular distribution heatmap (f) and local resolution estimation (g) for the consensus reconstruction of GATOR1-KICSTOR supercomplex. h-j, Masking and local refinement results of Nprl3-SZT2. Half-map FSC plots (h), angular particle distributions (i) and local resolution estimation (j) from local refinement on Nprl3-SZT2 regions. k, Composite map of GATOR1-KICSTOR supercomplex was generated for subsequent model building.

Extended Data Fig. 6 Model versus map FSC curves.

a-b, Map versus model FSC curves with masked and unmasked were calculated for full-length KICSTOR in state 1 and state 2 against the consensus full map in Phenix. c-e, Map versus model FSC curves with masked and unmasked were calculated for KICSTOR CCC in state 3, state 4 and state 5 against the consensus full map in Phenix. f, FSC between the model and map for the KICSTOR-GATOR1 complex against the cryo-EM map. g, Representative cryo-EM densities fitted to the model.

Extended Data Fig. 7 Structural comparison of GATOR1 in GAP mode and KICSTOR-bound conformations.

a, Structural alignment of GATOR1 in this study with the reported GAP-mode conformation (PDB 7T3B). Depdc5, Nprl2, and Nprl3 are colored in forest green/light-sky blue, dark salmon/yellow, and thistle/yellow-green, respectively, and presented in two different orientations. b, Close-up view of the Nprl3 longin-TINI domains (PDB 7T3B). c, Close-up view of the Nprl3 longin-TINI domains bound to SZT2NTD. d, Cryo-EM densities fitted to the Nprl3TINI model. e, The map covering the interface between Nprl3-SZT2. f, Structural alignment of Nprl2 with its GAP-mode conformation (PDB 7T3B). RMSD values for the superimposed structures are shown in Supplementary Table 2.

Extended Data Fig. 8 Hypothetical full-length KICSTOR model.

The model for residues 352-1713 of SZT2 was predicted from AlphaFold2 and then combined with the two refined models in this study.

Extended Data Fig. 9 Liposome-binding assays of different recombinant KICSTOR proteins with LUVs.

a, b,d, Liposome-binding assays of KICSTOR complex with LUVs of different compositions (a: 80% DOPC: 20% DOPE (neutral mix); b: 50% DOPC: 20% DOPE: 30% DOPS; d: 70% DOPC: 20% DOPE: 10% PtdIns3P). c, Quantification of different recombinant proteins to their interactions with LUVs containing 50% DOPC: 20% DOPE: 30% DOPS. f, g, i, Liposome-binding assays of KPTN/ITFG2/SZT2 complex with LUVs of different compositions (f: 80% DOPC: 20% DOPE (neutral mix); g: 50% DOPC: 20% DOPE: 30% DOPS; i: 70% DOPC: 20% DOPE: 10% PtdIns3P). k, l, n, Liposome-binding assays of GST-C12orf66 with LUVs of different compositions (k: 80% DOPC: 20% DOPE (neutral mix); l: 50% DOPC: 20% DOPE: 30% DOPS; n: 70% DOPC: 20% DOPE: 10% PtdIns3P). p, q, s, Liposome-binding assays of KPTN/ITFG2 complex with LUVs of different compositions (p: 80% DOPC: 20% DOPE (neutral mix); q: 50% DOPC: 20% DOPE: 30% DOPS; s: 70% DOPC: 20% DOPE: 10% PtdIns3P). e, Quantification of different recombinant proteins to their interactions with LUVs containing 70% DOPC: 20% DOPE: 10% PtdIns3P. h, m, r, Quantification of liposome-bound KPTN/ITFG2/SZT2 complex, GST-C12orf66, or KPTN/ITFG2 complex to LUVs containing DOPC and DOPE and additionally, the amount of DOPS, indicated in the figures. j, o, t, Quantification of liposome-bound KPTN/ITFG2/SZT2 complex, GST-C12orf66, or KPTN/ITFG2 complex to LUVs with different composition. Results were presented as mean ± s.e.m. Quantified data of liposome-binding assays in (c, e, h, j, m, o, r, t) represent mean ± s.e.m. of three independent experiments. % P was quantified as described in the Methods section. Statistical analysis was carried out using one-way analysis of variance (ANOVA). S, supernatant; P, pellet. The input lanes indicated 20% input of the proteins used in the experiment.

Source data

Extended Data Fig. 10 Hypothetical model of KICSTOR-GATOR1-GATOR2-Rag-Ragulator complex tethered on the lysosomal membrane.

a, Model was assembled using the reported structures of GATOR2 (PDB 7UHY), an AlphaFold2 prediction for WDR59 protein, and dual mode of GATOR1 which is bound to two copies of Rag-Ragulator, one in the “GAP conformation” and another one in the “inhibitory conformation” (PDB 7T3C) and KICSTOR from this study. b, Color code for the KICSTOR1, GATOR1, Rag-Ragulator and GATOR2 subunits. c, The reported structures of GATOR2 (PDB 7UHY), dual mode of GATOR1, which is bound to two copies of Rag-Ragulator, one in the “GAP conformation” and another one in the “inhibitory conformation” (PDB 7T3C), and an AlphaFold2 prediction for WDR59 protein, were assembled and superimposed on SEA complex (PDB 8ADL). The dotted line box indicates the interface between GATOR1 and GATOR2. d, KICSTOR from this study was superimposed on the GATOR1-dual mode. The dotted line box indicates the interface between GATOR1 and KICSTOR. e, Zoomed-in view on the clashing of SZT2SZN1-SZN2 and Sec13.

Supplementary information

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Supplementary Figs. 1–6, Tables 1–4 and source data for supplementary figures.

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Source Data Extended Data Fig. 4

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Source Data Extended Data Fig. 9

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Teng, F., Zeng, H., Mai, X. et al. Architecture of the human KICSTOR and GATOR1–KICSTOR complexes. Nat Struct Mol Biol 32, 2587–2600 (2025). https://doi.org/10.1038/s41594-025-01693-4

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