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- PDB-7ux2: cryo-EM structure of the Raptor-TFEB-Rag-Ragulator complex -

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Basic information

Entry
Database: PDB / ID: 7ux2
Titlecryo-EM structure of the Raptor-TFEB-Rag-Ragulator complex
Components
  • (Ragulator complex protein ...) x 5
  • (Ras-related GTP-binding protein ...) x 2
  • Regulatory-associated protein of mTOR
  • Transcription factor EB
KeywordsSIGNALING 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 / regulation of cholesterol efflux / Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / Ragulator complex / positive regulation of pentose-phosphate shunt / regulation of TORC1 signaling / antibacterial innate immune response ...regulation of cholesterol import / positive regulation of protein localization to lysosome / regulation of cell-substrate junction organization / regulation of cholesterol efflux / Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / Ragulator complex / positive regulation of pentose-phosphate shunt / regulation of TORC1 signaling / antibacterial innate immune response / positive regulation of odontoblast differentiation / regulation of lysosome organization / TORC1 complex / protein localization to lysosome / lysosome localization / MTOR signalling / cellular response to L-leucine / Energy dependent regulation of mTOR by LKB1-AMPK / endosome organization / Amino acids regulate mTORC1 / Dengue virus modulates apoptosis / positive regulation of osteoclast differentiation / regulation of TOR signaling / cellular response to osmotic stress / kinase activator activity / protein localization to membrane / Phosphorylated BMAL1:CLOCK (ARNTL:CLOCK) activates expression of core clock genes / lysosome organization / azurophil granule membrane / endosomal transport / regulation of cell size / Macroautophagy / humoral immune response / small GTPase-mediated signal transduction / RHOJ GTPase cycle / RHOQ GTPase cycle / embryonic placenta development / positive regulation of transcription by RNA polymerase III / TORC1 signaling / regulation of receptor recycling / CDC42 GTPase cycle / social behavior / response to amino acid / tertiary granule membrane / TOR signaling / RHOG GTPase cycle / RHOH GTPase cycle / mTORC1-mediated signalling / ficolin-1-rich granule membrane / RAC3 GTPase cycle / HSF1-dependent transactivation / RAC2 GTPase cycle / positive regulation of TOR signaling / positive regulation of lipid biosynthetic process / enzyme-substrate adaptor activity / protein kinase activator activity / protein serine/threonine kinase inhibitor activity / positive regulation of G1/S transition of mitotic cell cycle / specific granule membrane / RAC1 GTPase cycle / positive regulation of endothelial cell proliferation / cholesterol homeostasis / protein-membrane adaptor activity / 14-3-3 protein binding / positive regulation of TORC1 signaling / Transcriptional and post-translational regulation of MITF-M expression and activity / cellular response to nutrient levels / positive regulation of autophagy / positive regulation of glycolytic process / guanyl-nucleotide exchange factor activity / negative regulation of autophagy / RNA splicing / viral genome replication / cellular response to amino acid starvation / cellular response to starvation / Regulation of PTEN gene transcription / tumor necrosis factor-mediated signaling pathway / cellular response to amino acid stimulus / positive regulation of interleukin-8 production / regulation of autophagy / TP53 Regulates Metabolic Genes / cellular response to glucose stimulus / regulation of cell growth / phosphoprotein binding / positive regulation of protein localization to nucleus / MAP2K and MAPK activation / response to virus / small GTPase binding / autophagy / cytoplasmic stress granule / intracellular protein localization / sequence-specific double-stranded DNA binding / glucose homeostasis / GDP binding / late endosome / late endosome membrane / E3 ubiquitin ligases ubiquitinate target proteins / positive regulation of cell growth / GTPase binding / transcription regulator complex
Similarity search - Function
Transcription factor EB / Beta-Lactamase - #190 / MiT/TFE transcription factors, C-terminal / MiT/TFE transcription factors, N-terminal / Domain of unknown function (DUF3371) / MITF/TFEB/TFEC/TFE3 N-terminus / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR3 / Ragulator complex protein LAMTOR5 / Mitogen-activated protein kinase kinase 1 interacting ...Transcription factor EB / Beta-Lactamase - #190 / MiT/TFE transcription factors, C-terminal / MiT/TFE transcription factors, N-terminal / Domain of unknown function (DUF3371) / MITF/TFEB/TFEC/TFE3 N-terminus / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR3 / Ragulator complex protein LAMTOR5 / Mitogen-activated protein kinase kinase 1 interacting / Ragulator complex protein LAMTOR5 / Mitogen-activated protein kinase kinase 1 interacting / LAMTOR1/MEH1 / Late endosomal/lysosomal adaptor and MAPK and MTOR activator / Late endosomal/lysosomal adaptor and MAPK and MTOR activator / RagA/B / RagC/D / Gtr1/RagA G protein / Gtr1/RagA G protein conserved region / Raptor, N-terminal CASPase-like domain / Ragulator complex protein LAMTOR2-like / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / HEAT repeat / Roadblock/LAMTOR2 domain / Roadblock/LC7 domain / Roadblock/LC7 domain / HEAT repeat / Helix-loop-helix DNA-binding domain / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile. / Helix-loop-helix DNA-binding domain superfamily / Beta-Lactamase / Armadillo-like helical / WD domain, G-beta repeat / Armadillo-type fold / P-loop containing nucleotide triphosphate hydrolases / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Ragulator complex protein LAMTOR5 / Transcription factor EB / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR1 / Ras-related GTP-binding protein A / Regulatory-associated protein of mTOR / Ras-related GTP-binding protein C / Ragulator complex protein LAMTOR3 / Ragulator complex protein LAMTOR2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsCui, Z. / Hurley, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM111730 United States
CitationJournal: 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
DepositionMay 4, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Category: citation / citation_author
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.title / _citation.year
Revision 1.2Feb 8, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Mar 1, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Regulatory-associated protein of mTOR
B: Ras-related GTP-binding protein A
C: Ras-related GTP-binding protein C
D: Ragulator complex protein LAMTOR1
E: Ragulator complex protein LAMTOR2
F: Ragulator complex protein LAMTOR3
G: Ragulator complex protein LAMTOR4
H: Ragulator complex protein LAMTOR5
I: Ras-related GTP-binding protein A
J: Ras-related GTP-binding protein C
K: Ragulator complex protein LAMTOR1
L: Ragulator complex protein LAMTOR2
M: Ragulator complex protein LAMTOR3
N: Ragulator complex protein LAMTOR4
O: Ragulator complex protein LAMTOR5
P: Transcription factor EB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)496,49422
Polymers494,51316
Non-polymers1,9816
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AP

#1: Protein Regulatory-associated protein of mTOR / Raptor / p150 target of rapamycin (TOR)-scaffold 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
#9: Protein Transcription factor EB / Class E basic helix-loop-helix protein 35 / bHLHe35


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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P19484

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Ras-related GTP-binding protein ... , 2 types, 4 molecules BICJ

#2: Protein Ras-related GTP-binding protein A / Rag A / RagA / Adenovirus E3 14.7 kDa-interacting protein 1 / FIP-1


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
#3: Protein Ras-related GTP-binding protein C / RagC / GTPase-interacting protein 2 / TIB929


Mass: 44298.859 Da / Num. of mol.: 2 / Mutation: S75N
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 DKELFMGNHO

#4: Protein Ragulator complex protein LAMTOR1 / Late endosomal/lysosomal adaptor and MAPK and MTOR activator 1 / Lipid raft adaptor protein p18 / ...Late endosomal/lysosomal adaptor and MAPK and MTOR activator 1 / Lipid raft adaptor protein p18 / Protein associated with DRMs and endosomes / p27Kip1-releasing factor from RhoA / p27RF-Rho


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
#5: Protein Ragulator complex protein LAMTOR2 / Endosomal adaptor protein p14 / Late endosomal/lysosomal Mp1-interacting protein / Late ...Endosomal adaptor protein p14 / Late endosomal/lysosomal Mp1-interacting protein / Late endosomal/lysosomal adaptor and MAPK and MTOR activator 2 / Mitogen-activated protein-binding protein-interacting protein / MAPBP-interacting protein / Roadblock domain-containing protein 3


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
#6: Protein Ragulator complex protein LAMTOR3 / Late endosomal/lysosomal adaptor and MAPK and MTOR activator 3 / MEK-binding partner 1 / Mp1 / ...Late endosomal/lysosomal adaptor and MAPK and MTOR activator 3 / MEK-binding partner 1 / Mp1 / Mitogen-activated protein kinase kinase 1-interacting protein 1 / Mitogen-activated protein kinase scaffold protein 1


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
#7: Protein Ragulator complex protein LAMTOR4 / Late endosomal/lysosomal adaptor and MAPK and MTOR activator 4


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
#8: Protein Ragulator complex protein LAMTOR5 / Hepatitis B virus X-interacting protein / HBX-interacting protein / Late endosomal/lysosomal ...Hepatitis B virus X-interacting protein / HBX-interacting protein / Late endosomal/lysosomal adaptor and MAPK and MTOR activator 5


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 , 3 types, 6 molecules

#10: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#12: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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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 Raptor-TFEB-Rag-Ragulator complex / Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
4cryoSPARC3.2CTF correction
7UCSF ChimeraXmodel fitting
12cryoSPARC3.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 377569 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 34.77 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003527786
ELECTRON MICROSCOPYf_angle_d0.626237649
ELECTRON MICROSCOPYf_chiral_restr0.04324288
ELECTRON MICROSCOPYf_plane_restr0.00464820
ELECTRON MICROSCOPYf_dihedral_angle_d7.5043746

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