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- PDB-6nzd: Cryo-EM Structure of the Lysosomal Folliculin Complex (FLCN-FNIP2... -

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Basic information

Entry
Database: PDB / ID: 6nzd
TitleCryo-EM Structure of the Lysosomal Folliculin Complex (FLCN-FNIP2-RagA-RagC-Ragulator)
Components
  • (Ragulator complex protein ...) x 4
  • (Ras-related GTP-binding protein ...) x 2
  • Folliculin
  • Folliculin-interacting protein 2
  • Hepatitis B virus x interacting protein
Keywordssignaling protein/inhibitor / Lysosome / mTORC1 regulation / Amino acid sensing / GTPase / SIGNALING PROTEIN / signaling protein-inhibitor complex
Function / homology
Function and homology information


negative regulation of cell proliferation involved in kidney development / cell proliferation involved in kidney development / negative regulation of post-translational protein modification / 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 / FNIP-folliculin RagC/D GAP / Ragulator complex ...negative regulation of cell proliferation involved in kidney development / cell proliferation involved in kidney development / negative regulation of post-translational protein modification / 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 / FNIP-folliculin RagC/D GAP / Ragulator complex / regulation of Ras protein signal transduction / negative regulation of brown fat cell differentiation / protein localization to cell junction / regulation of TORC1 signaling / regulation of pro-B cell differentiation / negative regulation of lysosome organization / protein localization to lysosome / regulation of TOR signaling / Amino acids regulate mTORC1 / fibroblast migration / MTOR signalling / lysosome localization / endosome organization / Energy dependent regulation of mTOR by LKB1-AMPK / TORC1 signaling / ATPase inhibitor activity / cell-cell junction assembly / protein localization to membrane / kinase activator activity / negative regulation of glycolytic process / negative regulation of TOR signaling / negative regulation of cold-induced thermogenesis / endosomal transport / azurophil granule membrane / lysosome organization / small GTPase-mediated signal transduction / regulation of cell size / Macroautophagy / negative regulation of Rho protein signal transduction / positive regulation of transforming growth factor beta receptor signaling pathway / RHOJ GTPase cycle / RHOQ GTPase cycle / hemopoiesis / TOR signaling / regulation of protein phosphorylation / mTORC1-mediated signalling / tertiary granule membrane / CDC42 GTPase cycle / ficolin-1-rich granule membrane / RHOH GTPase cycle / enzyme inhibitor activity / RHOG GTPase cycle / regulation of receptor recycling / positive regulation of TOR signaling / RAC2 GTPase cycle / RAC3 GTPase cycle / enzyme-substrate adaptor activity / positive regulation of peptidyl-serine phosphorylation / response to amino acid / cellular response to nutrient levels / specific granule membrane / energy homeostasis / positive regulation of intrinsic apoptotic signaling pathway / protein-membrane adaptor activity / tumor necrosis factor-mediated signaling pathway / ERK1 and ERK2 cascade / RAC1 GTPase cycle / positive regulation of autophagy / positive regulation of TORC1 signaling / negative regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / negative regulation of autophagy / intrinsic apoptotic signaling pathway / GTPase activator activity / cellular response to amino acid starvation / transforming growth factor beta receptor signaling pathway / RNA splicing / viral genome replication / cholesterol homeostasis / guanyl-nucleotide exchange factor activity / cellular response to starvation / epithelial cell proliferation / Regulation of PTEN gene transcription / positive regulation of interleukin-8 production / TP53 Regulates Metabolic Genes / phosphoprotein binding / cellular response to amino acid stimulus / phosphatidylinositol 3-kinase/protein kinase B signal transduction / regulation of cell growth / positive regulation of protein-containing complex assembly / MAP2K and MAPK activation / negative regulation of ERK1 and ERK2 cascade / centriolar satellite / response to virus / positive regulation of protein localization to nucleus / intrinsic apoptotic signaling pathway in response to DNA damage / negative regulation of epithelial cell proliferation / mitotic spindle / GDP binding / late endosome membrane / late endosome
Similarity search - Function
Folliculin / Folliculin, DENN domain / Folliculin, DENN domain, C-terminal superfamily / Folliculin C-terminal domain / Folliculin-interacting protein family / Folliculin-interacting protein, N-terminal domain / Folliculin-interacting protein, middle domain / Folliculin-interacting protein, C-terminal domain / Folliculin-interacting protein N-terminus / Folliculin-interacting protein middle domain ...Folliculin / Folliculin, DENN domain / Folliculin, DENN domain, C-terminal superfamily / Folliculin C-terminal domain / Folliculin-interacting protein family / Folliculin-interacting protein, N-terminal domain / Folliculin-interacting protein, middle domain / Folliculin-interacting protein, C-terminal domain / Folliculin-interacting protein N-terminus / Folliculin-interacting protein middle domain / Folliculin-interacting protein C-terminus / Tripartite DENN domain, FNIP1/2-type / Tripartite DENN FNIP1/2-type domain profile. / Beta-Lactamase - #190 / Folliculin/SMCR8, longin domain / Folliculin/SMCR8, tripartite DENN domain / Vesicle coat protein involved in Golgi to plasma membrane transport / Tripartite DENN FLCN/SMCR8-type domain profile. / 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 / Gtr1/RagA G protein / RagC/D / Gtr1/RagA G protein conserved region / Ragulator complex protein LAMTOR2-like / Dynein light chain 2a, cytoplasmic / Roadblock/LAMTOR2 domain / Roadblock/LC7 domain / Roadblock/LC7 domain / Beta-Lactamase / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / Chem-L8S / Ragulator complex protein LAMTOR5 / Ragulator complex protein LAMTOR5 / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR1 / Ras-related GTP-binding protein A / Folliculin / Ras-related GTP-binding protein C / Folliculin-interacting protein 2 ...GUANOSINE-5'-DIPHOSPHATE / Chem-L8S / Ragulator complex protein LAMTOR5 / Ragulator complex protein LAMTOR5 / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR1 / Ras-related GTP-binding protein A / Folliculin / Ras-related GTP-binding protein C / Folliculin-interacting protein 2 / Ragulator complex protein LAMTOR3 / Ragulator complex protein LAMTOR2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsFromm, S.A. / Young, L.N. / Hurley, J.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)2R01GM111730-05 United States
CitationJournal: Science / Year: 2019
Title: Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex.
Authors: Rosalie E Lawrence / Simon A Fromm / Yangxue Fu / Adam L Yokom / Do Jin Kim / Ashley M Thelen / Lindsey N Young / Chun-Yan Lim / Avi J Samelson / James H Hurley / Roberto Zoncu /
Abstract: The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) ...The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagA:RagC GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo-electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.
History
DepositionFeb 13, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 4, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _pdbx_audit_support.funding_organization
Revision 1.4Nov 20, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
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 / _em_admin.last_update

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Structure visualization

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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: Hepatitis B virus x interacting protein
F: Ras-related GTP-binding protein A
G: Ras-related GTP-binding protein C
H: Folliculin
I: Folliculin-interacting protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)348,51611
Polymers347,5329
Non-polymers9832
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25470 Å2
ΔGint-169 kcal/mol
Surface area79660 Å2
MethodPISA

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Components

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Ragulator complex protein ... , 4 types, 4 molecules ABCD

#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

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Protein , 3 types, 3 molecules EHI

#5: Protein Hepatitis B virus x interacting protein / Ragulator complex protein LAMTOR5


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, UniProt: O43504*PLUS
#8: Protein Folliculin / BHD skin lesion fibrofolliculoma protein / Birt-Hogg-Dube syndrome protein


Mass: 69143.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FLCN, BHD / Cell line (production host): HEK 293 GNTI / Production host: Homo sapiens (human) / References: UniProt: Q8NFG4
#9: Protein Folliculin-interacting protein 2 / FNIP1-like protein / O6-methylguanine-induced apoptosis 1 protein


Mass: 122475.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FNIP2, FNIPL, KIAA1450, MAPO1 / Cell line (production host): HEK 293 GNTI / Production host: Homo sapiens (human) / References: UniProt: Q9P278

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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: 36615.168 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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Non-polymers , 2 types, 2 molecules

#10: 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
#11: 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

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Details

Has protein modificationY

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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: FLCN-FNIP2-RagA-RagC-Ragulator Complex / Type: COMPLEX / Details: RagA bound to GDP; RagC bound to XTPgammaS / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.34 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
2130 mMsodium chlorideNaCl1
32.5 mMmagnesium chlorideMgCl21
42 mMEGTA1
50.5 mMTCEP1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 W / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K / Details: Whatman 597

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 11 sec. / Electron dose: 65.6 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2703
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 44

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
9DireXmodel fitting
14cryoSPARC23D reconstruction
21PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 982343
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163376 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
16B9X

6b9x

A6B9X1
26B9X

6b9x

B6B9X1
36B9X

6b9x

C6B9X1
46B9X

6b9x

D6B9X1
56B9X

6b9x

E6B9X1
66EHR

6ehr

E6EHR248-68
76CES

6ces

A6CES3
86CES

6ces

C6CES3
93V42

3v42

B3V424
103LLU

3llu

A3LLU5119-139
RefinementHighest resolution: 3.6 Å

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