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- PDB-6wj2: CryoEM structure of the SLC38A9-RagA-RagC-Ragulator complex in th... -

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Basic information

Entry
Database: PDB / ID: 6wj2
TitleCryoEM structure of the SLC38A9-RagA-RagC-Ragulator complex in the pre-GAP state
Components
  • (Ragulator complex protein ...) x 5
  • (Ras-related GTP-binding protein ...) x 2
  • Sodium-coupled neutral amino acid transporter 9
KeywordsSIGNALING PROTEIN / small GTPase / mTORC1 activation / amino acid signaling / lysosome
Function / homology
Function and homology information


asparagine transport / L-asparagine transmembrane transporter activity / sterol sensor activity / L-arginine transmembrane transporter activity / L-arginine transmembrane transport / 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 ...asparagine transport / L-asparagine transmembrane transporter activity / sterol sensor activity / L-arginine transmembrane transporter activity / L-arginine transmembrane transport / 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 / L-glutamine transmembrane transporter activity / glutamine transport / FNIP-folliculin RagC/D GAP / Ragulator complex / protein localization to cell junction / amino acid transmembrane transport / L-leucine transmembrane transporter activity / L-amino acid transmembrane transporter activity / regulation of TORC1 signaling / protein localization to lysosome / regulation of TOR signaling / fibroblast migration / MTOR signalling / lysosome localization / Amino acids regulate mTORC1 / Energy dependent regulation of mTOR by LKB1-AMPK / endosome organization / TORC1 signaling / amino acid transmembrane transporter activity / kinase activator activity / arginine binding / protein localization to membrane / endosomal transport / azurophil granule membrane / lysosome organization / cholesterol binding / small GTPase-mediated signal transduction / Macroautophagy / regulation of cell size / RHOJ GTPase cycle / RHOQ GTPase cycle / mTORC1-mediated signalling / CDC42 GTPase cycle / tertiary granule membrane / RHOH GTPase cycle / RHOG GTPase cycle / ficolin-1-rich granule membrane / regulation of receptor recycling / RAC2 GTPase cycle / positive regulation of TOR signaling / RAC3 GTPase cycle / enzyme-substrate adaptor activity / response to amino acid / cellular response to nutrient levels / specific granule membrane / protein-membrane adaptor activity / RAC1 GTPase cycle / positive regulation of TORC1 signaling / negative regulation of autophagy / cellular response to amino acid starvation / RNA splicing / viral genome replication / cholesterol homeostasis / guanyl-nucleotide exchange factor activity / cellular response to starvation / Regulation of PTEN gene transcription / tumor necrosis factor-mediated signaling pathway / positive regulation of interleukin-8 production / TP53 Regulates Metabolic Genes / cellular response to amino acid stimulus / phosphoprotein binding / regulation of cell growth / MAP2K and MAPK activation / positive regulation of protein localization to nucleus / response to virus / GDP binding / late endosome membrane / E3 ubiquitin ligases ubiquitinate target proteins / intracellular protein localization / late endosome / glucose homeostasis / GTPase binding / molecular adaptor activity / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / endosome membrane / lysosome / positive regulation of canonical NF-kappaB signal transduction / positive regulation of MAPK cascade / intracellular signal transduction / membrane raft / protein heterodimerization activity / lysosomal membrane / focal adhesion / intracellular membrane-bounded organelle / GTPase activity / ubiquitin protein ligase binding / apoptotic process / DNA-templated transcription / Neutrophil degranulation / positive regulation of gene expression
Similarity search - Function
Amino acid transporter, transmembrane domain / Transmembrane amino acid transporter protein / LAMTOR1/MEH1 / Late endosomal/lysosomal adaptor and MAPK and MTOR activator / Late endosomal/lysosomal adaptor and MAPK and MTOR activator / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR3 / Ragulator complex protein LAMTOR5 / RagA/B / Mitogen-activated protein kinase kinase 1 interacting ...Amino acid transporter, transmembrane domain / Transmembrane amino acid transporter protein / LAMTOR1/MEH1 / Late endosomal/lysosomal adaptor and MAPK and MTOR activator / Late endosomal/lysosomal adaptor and MAPK and MTOR activator / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR3 / Ragulator complex protein LAMTOR5 / RagA/B / Mitogen-activated protein kinase kinase 1 interacting / Ragulator complex protein LAMTOR5 / Mitogen-activated protein kinase kinase 1 interacting / RagC/D / Gtr1/RagA G protein / Gtr1/RagA G protein conserved region / Ragulator complex protein LAMTOR2-like / Roadblock/LAMTOR2 domain / Roadblock/LC7 domain / Roadblock/LC7 domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / Chem-L8S / Ragulator complex protein LAMTOR5 / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR1 / Ras-related GTP-binding protein A / Neutral amino acid transporter 9 / 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: 3.2 Å
AuthorsFromm, S.A. / Hurley, J.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM111730 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9.
Authors: Simon A Fromm / Rosalie E Lawrence / James H Hurley /
Abstract: The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin ...The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator, and the FLCN:FNIP2 (FLCN-interacting protein 2) GTPase activating protein (GAP) complex, and prevents Rag dimer activation during amino acid starvation. How the LFC is disassembled upon amino acid refeeding is an outstanding question. Here we show that the cytoplasmic tail of the human lysosomal solute carrier family 38 member 9 (SLC38A9) destabilizes the LFC and thereby triggers GAP activity of FLCN:FNIP2 toward RagC. We present the cryo-EM structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre- and post-GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.
History
DepositionApr 11, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Nov 18, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Ragulator complex protein LAMTOR1
B: Ragulator complex protein LAMTOR2
C: Ragulator complex protein LAMTOR3
D: Ragulator complex protein LAMTOR4
E: Ragulator complex protein LAMTOR5
F: Ras-related GTP-binding protein A
G: Ras-related GTP-binding protein C
H: Sodium-coupled neutral amino acid transporter 9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)173,27911
Polymers172,2718
Non-polymers1,0083
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21100 Å2
ΔGint-171 kcal/mol
Surface area55810 Å2

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Components

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Ragulator complex protein ... , 5 types, 5 molecules ABCDE

#1: 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: 18325.350 Da / Num. of mol.: 1 / Mutation: G2A
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
#2: 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: 13645.579 Da / Num. of mol.: 1
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
#3: 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.: 1
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
#4: Protein Ragulator complex protein LAMTOR4 / Late endosomal/lysosomal adaptor and MAPK and MTOR activator 4


Mass: 10753.236 Da / Num. of mol.: 1
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
#5: Protein Ragulator complex protein LAMTOR5 / Hepatitis B virus x interacting protein


Mass: 18178.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR5, HBXIP, hCG_40252 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0C4DGV4

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Ras-related GTP-binding protein ... , 2 types, 2 molecules FG

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


Mass: 39362.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7L523
#7: Protein Ras-related GTP-binding protein C / RagC / GTPase-interacting protein 2 / TIB929


Mass: 44758.336 Da / Num. of mol.: 1 / Mutation: D181N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9HB90

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Protein , 1 types, 1 molecules H

#8: Protein Sodium-coupled neutral amino acid transporter 9 / Solute carrier family 38 member 9 / Up-regulated in lung cancer 11


Mass: 13610.139 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC38A9, URLC11 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8NBW4

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Non-polymers , 3 types, 3 molecules

#9: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#10: Chemical ChemComp-L8S / 9-{5-O-[(S)-hydroxy{[(R)-hydroxy(thiophosphonooxy)phosphoryl]oxy}phosphoryl]-alpha-L-arabinofuranosyl}-3,9-dihydro-1H-purine-2,6-dione


Mass: 540.231 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N4O14P3S
#11: 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

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of pentameric Ragulator, dimeric Rag GTPases and SLC38A9COMPLEXRagA bound to GDP, RagC bound to XTPgammaS#1-#80MULTIPLE SOURCES
2pentameric RagulatorCOMPLEX#1-#51RECOMBINANT
3dimeric Rag GTPasesCOMPLEX#6-#71RECOMBINANT
4SLC38A9COMPLEX#81RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Spodoptera frugiperda (fall armyworm)7108
23Spodoptera frugiperda (fall armyworm)7108
34Homo sapiens (human)9606HEK293
Buffer solutionpH: 7.4
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 magnification: 29000 X / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.1 sec. / Electron dose: 59 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: dev_3736: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1.13CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
13cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1419155
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 129553 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 6NZD / Initial refinement model-ID: 1 / PDB-ID: 6NZD

6nzd

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
3C
4D
5E
6F
7G
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029064
ELECTRON MICROSCOPYf_angle_d0.43712303
ELECTRON MICROSCOPYf_dihedral_angle_d14.7791247
ELECTRON MICROSCOPYf_chiral_restr0.041447
ELECTRON MICROSCOPYf_plane_restr0.0031568

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