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- PDB-9ed8: Intermediate state of mTOR on membrane -

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Basic information

Entry
Database: PDB / ID: 9ed8
TitleIntermediate state of mTOR on membrane
Components
  • GTP-binding protein Rheb
  • Serine/threonine-protein kinase mTOR
  • Target of rapamycin complex subunit LST8
KeywordsSIGNALING PROTEIN / mTORC1 / cell growth / membrane
Function / homology
Function and homology information


regulation of type B pancreatic cell development / RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / regulation of locomotor rhythm / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / positive regulation of wound healing, spreading of epidermal cells / TORC2 signaling / TORC2 complex ...regulation of type B pancreatic cell development / RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / regulation of locomotor rhythm / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / positive regulation of wound healing, spreading of epidermal cells / TORC2 signaling / TORC2 complex / regulation of membrane permeability / cellular response to leucine starvation / negative regulation of lysosome organization / heart valve morphogenesis / TFIIIC-class transcription factor complex binding / TORC1 complex / voluntary musculoskeletal movement / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / calcineurin-NFAT signaling cascade / RNA polymerase III type 3 promoter sequence-specific DNA binding / positive regulation of keratinocyte migration / regulation of osteoclast differentiation / regulation of lysosome organization / MTOR signalling / cellular response to nutrient / cellular response to L-leucine / energy reserve metabolic process / Amino acids regulate mTORC1 / regulation of autophagosome assembly / Energy dependent regulation of mTOR by LKB1-AMPK / TORC1 signaling / ruffle organization / serine/threonine protein kinase complex / cellular response to methionine / negative regulation of cell size / positive regulation of ubiquitin-dependent protein catabolic process / cellular response to osmotic stress / negative regulation of protein localization to nucleus / anoikis / inositol hexakisphosphate binding / cardiac muscle cell development / negative regulation of calcineurin-NFAT signaling cascade / negative regulation of cold-induced thermogenesis / regulation of myelination / positive regulation of transcription by RNA polymerase III / small GTPase-mediated signal transduction / negative regulation of macroautophagy / Macroautophagy / positive regulation of myotube differentiation / regulation of cell size / Constitutive Signaling by AKT1 E17K in Cancer / positive regulation of actin filament polymerization / germ cell development / TOR signaling / behavioral response to pain / mTORC1-mediated signalling / oligodendrocyte differentiation / positive regulation of oligodendrocyte differentiation / positive regulation of translational initiation / protein kinase activator activity / CD28 dependent PI3K/Akt signaling / HSF1-dependent transactivation / positive regulation of TOR signaling / regulation of macroautophagy / response to amino acid / 'de novo' pyrimidine nucleobase biosynthetic process / positive regulation of epithelial to mesenchymal transition / vascular endothelial cell response to laminar fluid shear stress / positive regulation of lipid biosynthetic process / heart morphogenesis / cellular response to nutrient levels / neuronal action potential / regulation of cellular response to heat / positive regulation of lamellipodium assembly / cardiac muscle contraction / phagocytic vesicle / T cell costimulation / positive regulation of stress fiber assembly / cytoskeleton organization / positive regulation of TORC1 signaling / endomembrane system / negative regulation of insulin receptor signaling pathway / negative regulation of autophagy / cellular response to amino acid starvation / positive regulation of translation / regulation of signal transduction by p53 class mediator / positive regulation of glycolytic process / cellular response to starvation / protein serine/threonine kinase activator activity / Regulation of PTEN gene transcription / VEGFR2 mediated vascular permeability / post-embryonic development / TP53 Regulates Metabolic Genes / regulation of actin cytoskeleton organization / spliceosomal complex / cellular response to amino acid stimulus / non-specific protein-tyrosine kinase / macroautophagy / phosphoprotein binding / response to nutrient levels
Similarity search - Function
Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3385, target of rapamycin protein / Serine/threonine-protein kinase mTOR domain / Domain of unknown function / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR / FKBP12-rapamycin binding domain superfamily / FKBP12-rapamycin binding domain / Rapamycin binding domain / Serine/threonine-protein kinase ATR-like, HEAT repeats ...Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3385, target of rapamycin protein / Serine/threonine-protein kinase mTOR domain / Domain of unknown function / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR / FKBP12-rapamycin binding domain superfamily / FKBP12-rapamycin binding domain / Rapamycin binding domain / Serine/threonine-protein kinase ATR-like, HEAT repeats / : / FATC domain / PIK-related kinase, FAT / FAT domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Quinoprotein alcohol dehydrogenase-like superfamily / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Small GTPase, Ras-type / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Small GTPase Ras domain profile. / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Tetratricopeptide-like helical domain superfamily / WD domain, G-beta repeat / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / Serine/threonine-protein kinase mTOR / GTP-binding protein Rheb / Target of rapamycin complex subunit LST8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.61 Å
AuthorsCui, Z. / Hurley, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA285366 United States
CitationJournal: Nature / Year: 2025
Title: Structural basis for mTORC1 activation on the lysosomal membrane.
Authors: Zhicheng Cui / Alessandra Esposito / Gennaro Napolitano / Andrea Ballabio / James H Hurley /
Abstract: The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth factor (GF) and nutrient signals to stimulate anabolic processes connected to cell growth and inhibit catabolic processes such ...The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth factor (GF) and nutrient signals to stimulate anabolic processes connected to cell growth and inhibit catabolic processes such as autophagy. GF signalling through the tuberous sclerosis complex regulates the lysosomally localized small GTPase RAS homologue enriched in brain (RHEB). Direct binding of RHEB-GTP to the mTOR kinase subunit of mTORC1 allosterically activates the kinase by inducing a large-scale conformational change. Here we reconstituted mTORC1 activation on membranes by RHEB, RAGs and Ragulator. Cryo-electron microscopy showed that RAPTOR and mTOR interact directly with the membrane. Full engagement of the membrane anchors is required for optimal alignment of the catalytic residues in the mTOR kinase active site. Converging signals from GFs and nutrients drive mTORC1 recruitment to and activation on lysosomal membrane in a four-step process, consisting of (1) RAG-Ragulator-driven recruitment to within ~100 Å of the lysosomal membrane; (2) RHEB-driven recruitment to within ~40 Å; (3) RAPTOR-membrane engagement and intermediate enzyme activation; and (4) mTOR-membrane engagement and full enzyme activation. RHEB and membrane engagement combined leads to full catalytic activation and structurally explains GF and nutrient signal integration at the lysosome.
History
DepositionNov 16, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 10, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 1, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Serine/threonine-protein kinase mTOR
B: Target of rapamycin complex subunit LST8
L: GTP-binding protein Rheb
hetero molecules


Theoretical massNumber of molelcules
Total (without water)347,4177
Polymers345,6883
Non-polymers1,7304
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ABL

#1: Protein Serine/threonine-protein kinase mTOR / FK506-binding protein 12-rapamycin complex-associated protein 1 / FKBP12-rapamycin complex- ...FK506-binding protein 12-rapamycin complex-associated protein 1 / FKBP12-rapamycin complex-associated protein / Mammalian target of rapamycin / mTOR / Mechanistic target of rapamycin / Rapamycin and FKBP12 target 1 / Rapamycin target protein 1


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) / Strain (production host): HEK 293F gnti-
References: UniProt: P42345, non-specific serine/threonine protein kinase
#2: Protein Target of rapamycin complex subunit LST8 / TORC subunit LST8 / G protein beta subunit-like / Protein GbetaL / Mammalian lethal with SEC13 ...TORC subunit LST8 / G protein beta subunit-like / Protein GbetaL / Mammalian lethal with SEC13 protein 8 / mLST8


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) / Strain (production host): HEK 293F gnti- / References: UniProt: Q9BVC4
#3: Protein GTP-binding protein Rheb / Ras homolog enriched in brain


Mass: 20519.449 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RHEB, RHEB2 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2
References: UniProt: Q15382, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6
#6: Chemical ChemComp-GSP / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE


Mass: 539.246 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3S
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The mTORC1-Rag-Ragulator-4EBP1 complex on membrane / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK 293F gnti-
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
4cryoSPARC4.4CTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
11ISOLDEmodel refinement
15cryoSPARC4.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109105 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 141.89 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00422565
ELECTRON MICROSCOPYf_angle_d0.672830589
ELECTRON MICROSCOPYf_chiral_restr0.04033451
ELECTRON MICROSCOPYf_plane_restr0.0053894
ELECTRON MICROSCOPYf_dihedral_angle_d8.36083071

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