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Yorodumi- PDB-7uxc: cryo-EM structure of the mTORC1-TFEB-Rag-Ragulator complex with s... -
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-Basic information
Entry | Database: PDB / ID: 7uxc | ||||||
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Title | cryo-EM structure of the mTORC1-TFEB-Rag-Ragulator complex with symmetry expansion | ||||||
Components |
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Keywords | SIGNALING PROTEIN / mTORC1 / TFEB / Lysosome biogenesis / Autophagy | ||||||
Function / homology | Function and homology information regulation of cholesterol import / positive regulation of protein localization to lysosome / regulation of cell-substrate junction organization / Gtr1-Gtr2 GTPase complex / regulation of cholesterol efflux / positive regulation of RNA polymerase II regulatory region sequence-specific DNA binding / FNIP-folliculin RagC/D GAP / Ragulator complex / protein localization to cell junction / positive regulation of cytoplasmic translational initiation ...regulation of cholesterol import / positive regulation of protein localization to lysosome / regulation of cell-substrate junction organization / Gtr1-Gtr2 GTPase complex / regulation of cholesterol efflux / positive regulation of RNA polymerase II regulatory region sequence-specific DNA binding / FNIP-folliculin RagC/D GAP / Ragulator complex / protein localization to cell junction / positive regulation of cytoplasmic translational initiation / positive regulation of pentose-phosphate shunt / RNA polymerase III type 1 promoter sequence-specific DNA binding / RNA polymerase III type 2 promoter sequence-specific DNA binding / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / antibacterial innate immune response / cellular response to leucine starvation / regulation of TORC1 signaling / regulation of membrane permeability / heart valve morphogenesis / TFIIIC-class transcription factor complex binding / negative regulation of lysosome organization / RNA polymerase III type 3 promoter sequence-specific DNA binding / TORC2 complex / TORC1 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / protein localization to lysosome / regulation of autophagosome assembly / calcineurin-NFAT signaling cascade / nucleus localization / TORC1 signaling / voluntary musculoskeletal movement / positive regulation of odontoblast differentiation / regulation of osteoclast differentiation / regulation of TOR signaling / positive regulation of keratinocyte migration / endosome organization / cellular response to L-leucine / MTOR signalling / Amino acids regulate mTORC1 / fibroblast migration / cellular response to nutrient / lysosome localization / energy reserve metabolic process / Energy dependent regulation of mTOR by LKB1-AMPK / negative regulation of cell size / ruffle organization / protein serine/threonine kinase inhibitor activity / protein localization to membrane / kinase activator activity / positive regulation of osteoclast differentiation / cellular response to osmotic stress / enzyme-substrate adaptor activity / negative regulation of protein localization to nucleus / anoikis / cardiac muscle cell development / positive regulation of transcription by RNA polymerase III / negative regulation of calcineurin-NFAT signaling cascade / regulation of myelination / azurophil granule membrane / endosomal transport / regulation of cell size / Macroautophagy / positive regulation of oligodendrocyte differentiation / small GTPase-mediated signal transduction / negative regulation of macroautophagy / lysosome organization / positive regulation of actin filament polymerization / positive regulation of myotube differentiation / protein kinase activator activity / RHOJ GTPase cycle / behavioral response to pain / RHOQ GTPase cycle / oligodendrocyte differentiation / mTORC1-mediated signalling / Constitutive Signaling by AKT1 E17K in Cancer / germ cell development / CD28 dependent PI3K/Akt signaling / cellular response to nutrient levels / positive regulation of phosphoprotein phosphatase activity / social behavior / tertiary granule membrane / CDC42 GTPase cycle / RHOH GTPase cycle / humoral immune response / ficolin-1-rich granule membrane / HSF1-dependent transactivation / RHOG GTPase cycle / TOR signaling / positive regulation of TOR signaling / regulation of receptor recycling / neuronal action potential / RAC2 GTPase cycle / embryonic placenta development / RAC3 GTPase cycle / positive regulation of translational initiation / regulation of macroautophagy / response to amino acid / positive regulation of G1/S transition of mitotic cell cycle Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
Authors | Cui, Z. / Hurley, J. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nature / Year: 2023 Title: Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex. Authors: Zhicheng Cui / Gennaro Napolitano / Mariana E G de Araujo / Alessandra Esposito / Jlenia Monfregola / Lukas A Huber / Andrea Ballabio / James H Hurley / Abstract: The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its ...The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagC-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7uxc.cif.gz | 2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7uxc.ent.gz | 1.6 MB | Display | PDB format |
PDBx/mmJSON format | 7uxc.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7uxc_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 7uxc_full_validation.pdf.gz | 1.7 MB | Display | |
Data in XML | 7uxc_validation.xml.gz | 160.5 KB | Display | |
Data in CIF | 7uxc_validation.cif.gz | 247.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ux/7uxc ftp://data.pdbj.org/pub/pdb/validation_reports/ux/7uxc | HTTPS FTP |
-Related structure data
Related structure data | 26857MC 7ux2C 7uxhC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 4 types, 4 molecules ABCR
#1: Protein | Mass: 289257.969 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MTOR, FRAP, FRAP1, FRAP2, RAFT1, RAPT1 / Production host: Homo sapiens (human) References: UniProt: P42345, non-specific serine/threonine protein kinase |
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#2: Protein | Mass: 35910.090 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MLST8, GBL, LST8 / Production host: Homo sapiens (human) / References: UniProt: Q9BVC4 |
#3: Protein | Mass: 149200.016 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RPTOR, KIAA1303, RAPTOR / Production host: Homo sapiens (human) / References: UniProt: Q8N122 |
#11: Protein | Mass: 52926.621 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: TFEB, BHLHE35 / Production host: Homo sapiens (human) / References: UniProt: P19484 |
-Ras-related GTP-binding protein ... , 2 types, 4 molecules DKEL
#4: Protein | Mass: 36600.195 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGA / Production host: Homo sapiens (human) References: UniProt: Q7L523, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement #5: Protein | Mass: 44298.859 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGC / Production host: Homo sapiens (human) / References: UniProt: Q9HB90 |
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-Ragulator complex protein ... , 5 types, 10 molecules FMGNHOIPJQ
#6: Protein | Mass: 17762.775 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR1, C11orf59, PDRO, PP7157 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6IAA8 #7: Protein | Mass: 13517.450 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR2, MAPBPIP, ROBLD3, HSPC003 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9Y2Q5 #8: Protein | Mass: 13637.678 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR3, MAP2K1IP1, MAPKSP1, PRO2783 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9UHA4 #9: Protein | Mass: 10753.236 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR4, C7orf59 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q0VGL1 #10: Protein | Mass: 9622.900 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR5, HBXIP, XIP / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O43504 |
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-Non-polymers , 4 types, 7 molecules
#12: Chemical | ChemComp-IHP / | ||||
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#13: Chemical | #14: Chemical | #15: Chemical | |
-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: The mTORC1-TFEB-Rag-Ragulator complex / Type: COMPLEX / Entity ID: #1-#11 / Source: MULTIPLE SOURCES |
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Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192332 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 96.35 Å2 | ||||||||||||||||||||||||||||||||
Refine LS restraints |
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