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- PDB-6ulg: Cryo-EM structure of the FLCN-FNIP2-Rag-Ragulator complex -

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Entry
Database: PDB / ID: 6ulg
TitleCryo-EM structure of the FLCN-FNIP2-Rag-Ragulator complex
Components
  • (Ragulator complex protein ...) x 5
  • (Ras-related GTP-binding protein ...) x 2
  • Folliculin
  • Folliculin-interacting protein 2
KeywordsSIGNALING PROTEIN / FLCN-FNIP2 / Rag GTPases / Ragulator
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 / regulation of cholesterol efflux / Gtr1-Gtr2 GTPase complex / 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 / regulation of cholesterol efflux / Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / Ragulator complex / regulation of Ras protein signal transduction / protein localization to cell junction / regulation of pro-B cell differentiation / regulation of TORC1 signaling / negative regulation of lysosome organization / protein localization to lysosome / fibroblast migration / lysosome localization / MTOR signalling / ATPase inhibitor activity / regulation of TOR signaling / endosome organization / Energy dependent regulation of mTOR by LKB1-AMPK / Amino acids regulate mTORC1 / negative regulation of brown fat cell differentiation / cell-cell junction assembly / negative regulation of TOR signaling / kinase activator activity / negative regulation of cold-induced thermogenesis / negative regulation of Rho protein signal transduction / protein localization to membrane / negative regulation of glycolytic process / endosomal transport / azurophil granule membrane / regulation of cell size / small GTPase-mediated signal transduction / lysosome organization / Macroautophagy / RHOJ GTPase cycle / positive regulation of transforming growth factor beta receptor signaling pathway / RHOQ GTPase cycle / hemopoiesis / CDC42 GTPase cycle / TORC1 signaling / tertiary granule membrane / RHOG GTPase cycle / mTORC1-mediated signalling / TOR signaling / response to amino acid / ficolin-1-rich granule membrane / RHOH GTPase cycle / regulation of receptor recycling / RAC3 GTPase cycle / RAC2 GTPase cycle / positive regulation of TOR signaling / enzyme-substrate adaptor activity / specific granule membrane / ERK1 and ERK2 cascade / positive regulation of intrinsic apoptotic signaling pathway / energy homeostasis / RAC1 GTPase cycle / protein-membrane adaptor activity / positive regulation of TORC1 signaling / transforming growth factor beta receptor signaling pathway / positive regulation of autophagy / epithelial cell proliferation / intrinsic apoptotic signaling pathway / guanyl-nucleotide exchange factor activity / cellular response to nutrient levels / RNA splicing / enzyme inhibitor activity / viral genome replication / cholesterol homeostasis / cellular response to amino acid starvation / negative regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / Regulation of PTEN gene transcription / cellular response to starvation / tumor necrosis factor-mediated signaling pathway / negative regulation of autophagy / GTPase activator activity / positive regulation of interleukin-8 production / TP53 Regulates Metabolic Genes / phosphatidylinositol 3-kinase/protein kinase B signal transduction / cellular response to amino acid stimulus / positive regulation of protein-containing complex assembly / phosphoprotein binding / regulation of cell growth / MAP2K and MAPK activation / negative regulation of ERK1 and ERK2 cascade / positive regulation of protein localization to nucleus / intrinsic apoptotic signaling pathway in response to DNA damage / negative regulation of epithelial cell proliferation / response to virus / centriolar satellite / mitotic spindle / GDP binding / intracellular protein localization / late endosome / late endosome membrane / glucose homeostasis
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. / 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 / Ragulator complex protein LAMTOR2-like / Dynein light chain 2a, cytoplasmic / Roadblock/LAMTOR2 domain / Roadblock/LC7 domain / Roadblock/LC7 domain / Beta-Lactamase / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / 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: 3.31 Å
AuthorsShen, K. / Rogala, K.B. / Yu, Z.H. / Sabatini, D.M.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA103866 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA129105 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R37 AI47389 United States
Department of Defense (DOD, United States)W81XWH-15-1-0230 United States
Lustgarten Foundation United States
Howard Hughes Medical Institute (HHMI) United States
Tuberous Sclerosis Association United States
CitationJournal: Cell / Year: 2019
Title: Cryo-EM Structure of the Human FLCN-FNIP2-Rag-Ragulator Complex.
Authors: Kuang Shen / Kacper B Rogala / Hui-Ting Chou / Rick K Huang / Zhiheng Yu / David M Sabatini /
Abstract: mTORC1 controls anabolic and catabolic processes in response to nutrients through the Rag GTPase heterodimer, which is regulated by multiple upstream protein complexes. One such regulator, FLCN- ...mTORC1 controls anabolic and catabolic processes in response to nutrients through the Rag GTPase heterodimer, which is regulated by multiple upstream protein complexes. One such regulator, FLCN-FNIP2, is a GTPase activating protein (GAP) for RagC/D, but despite its important role, how it activates the Rag GTPase heterodimer remains unknown. We used cryo-EM to determine the structure of FLCN-FNIP2 in a complex with the Rag GTPases and Ragulator. FLCN-FNIP2 adopts an extended conformation with two pairs of heterodimerized domains. The Longin domains heterodimerize and contact both nucleotide binding domains of the Rag heterodimer, while the DENN domains interact at the distal end of the structure. Biochemical analyses reveal a conserved arginine on FLCN as the catalytic arginine finger and lead us to interpret our structure as an on-pathway intermediate. These data reveal features of a GAP-GTPase interaction and the structure of a critical component of the nutrient-sensing mTORC1 pathway.
History
DepositionOct 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.0Nov 20, 2019Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Nov 20, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Nov 20, 2019Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Nov 20, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Nov 20, 2019Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Nov 20, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Dec 11, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Dec 25, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.3May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
L: Folliculin
A: Ragulator complex protein LAMTOR3
B: Ragulator complex protein LAMTOR2
C: Ragulator complex protein LAMTOR5
D: Ragulator complex protein LAMTOR4
E: Ragulator complex protein LAMTOR1
F: Ras-related GTP-binding protein A
G: Ras-related GTP-binding protein C
N: Folliculin-interacting protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)333,99512
Polymers333,0059
Non-polymers9903
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules LN

#1: Protein Folliculin / BHD skin lesion fibrofolliculoma protein / Birt-Hogg-Dube syndrome protein


Mass: 64551.191 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FLCN, BHD / Production host: Homo sapiens (human) / References: UniProt: Q8NFG4
#9: Protein Folliculin-interacting protein 2 / FNIP1-like protein / O6-methylguanine-induced apoptosis 1 protein


Mass: 122260.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FNIP2, FNIPL, KIAA1450, MAPO1 / Production host: Homo sapiens (human) / References: UniProt: Q9P278

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

#2: 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: Escherichia coli (E. coli) / References: UniProt: Q9UHA4
#3: 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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR2, MAPBPIP, ROBLD3, HSPC003 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y2Q5
#4: 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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR5, HBXIP, XIP / Production host: Escherichia coli (E. coli) / References: UniProt: O43504
#5: 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: Escherichia coli (E. coli) / References: UniProt: Q0VGL1
#6: 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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LAMTOR1, C11orf59, PDRO, PP7157 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6IAA8

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

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


Mass: 36628.168 Da / Num. of mol.: 1 / Mutation: T21N
Source method: isolated from a genetically manipulated source
Details: RagA containing a T21N mutation / Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGA / Production host: Escherichia coli (E. coli) / References: UniProt: Q7L523
#8: Protein Ras-related GTP-binding protein C / RagC / GTPase-interacting protein 2 / TIB929


Mass: 44271.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGC / Production host: Escherichia coli (E. coli) / References: UniProt: Q9HB90

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Non-polymers , 3 types, 3 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-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM
#12: 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 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: Nonameric complex of FLCN-FNIP2 with its substrate Rag GTPases and the scaffolding protein complex Ragulator
Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightValue: 0.38 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 8 mg/ml / Embedding 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 59.2 e/Å2 / Film or detector model: GATAN K2 BASE (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126984 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00616668
ELECTRON MICROSCOPYf_angle_d0.90822543
ELECTRON MICROSCOPYf_dihedral_angle_d10.06610438
ELECTRON MICROSCOPYf_chiral_restr0.0542577
ELECTRON MICROSCOPYf_plane_restr0.0062856

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