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- PDB-6ces: Cryo-EM structure of GATOR1-RAG -

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Basic information

Entry
Database: PDB / ID: 6ces
TitleCryo-EM structure of GATOR1-RAG
Components
  • (GATOR complex protein ...) x 3
  • (Ras-related GTP-binding protein ...) x 2
KeywordsSIGNALING PROTEIN / mTORC1 amino-acid sensing lysosome growth control
Function / homology
Function and homology information


GATOR1 complex / Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / aorta morphogenesis / regulation of TORC1 signaling / protein localization to lysosome / TORC1 signaling / regulation of TOR signaling / MTOR signalling / Amino acids regulate mTORC1 ...GATOR1 complex / Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / aorta morphogenesis / regulation of TORC1 signaling / protein localization to lysosome / TORC1 signaling / regulation of TOR signaling / MTOR signalling / Amino acids regulate mTORC1 / Energy dependent regulation of mTOR by LKB1-AMPK / protein localization to membrane / enzyme-substrate adaptor activity / cardiac muscle tissue development / negative regulation of TOR signaling / vacuolar membrane / ventricular septum development / negative regulation of kinase activity / Macroautophagy / small GTPase-mediated signal transduction / roof of mouth development / mTORC1-mediated signalling / cellular response to nutrient levels / positive regulation of TOR signaling / response to amino acid / positive regulation of autophagy / protein-membrane adaptor activity / negative regulation of TORC1 signaling / positive regulation of TORC1 signaling / tumor necrosis factor-mediated signaling pathway / cellular response to amino acid starvation / cellular response to starvation / negative regulation of autophagy / RNA splicing / GTPase activator activity / Regulation of PTEN gene transcription / TP53 Regulates Metabolic Genes / phosphoprotein binding / cellular response to amino acid stimulus / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / protein localization / small GTPase binding / GDP binding / E3 ubiquitin ligases ubiquitinate target proteins / GTPase binding / glucose homeostasis / molecular adaptor activity / lysosome / intracellular signal transduction / protein heterodimerization activity / lysosomal membrane / intracellular membrane-bounded organelle / GTPase activity / DNA-templated transcription / apoptotic process / ubiquitin protein ligase binding / protein-containing complex binding / GTP binding / perinuclear region of cytoplasm / magnesium ion binding / protein homodimerization activity / nucleoplasm / nucleus / cytoplasm / cytosol
Similarity search - Function
Beta-Lactamase - #190 / : / Nitrogen permease regulator 3 / Nitrogen permease regulator 2 / Vacuolar membrane-associated protein Iml1 / DEPDC5 protein, C-terminal / Nitrogen Permease regulator of amino acid transport activity 3 / Nitrogen permease regulator 2 / Vacuolar membrane-associated protein Iml1, N-terminal domain / DEPDC5 protein C-terminal region ...Beta-Lactamase - #190 / : / Nitrogen permease regulator 3 / Nitrogen permease regulator 2 / Vacuolar membrane-associated protein Iml1 / DEPDC5 protein, C-terminal / Nitrogen Permease regulator of amino acid transport activity 3 / Nitrogen permease regulator 2 / Vacuolar membrane-associated protein Iml1, N-terminal domain / DEPDC5 protein C-terminal region / RagA/B / Gtr1/RagA G protein / RagC/D / Gtr1/RagA G protein conserved region / Domain found in Dishevelled, Egl-10, and Pleckstrin (DEP) / DEP domain profile. / Domain found in Dishevelled, Egl-10, and Pleckstrin / DEP domain / Beta-Lactamase / P-loop containing nucleotide triphosphate hydrolases / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / GATOR1 complex protein DEPDC5 / GATOR1 complex protein NPRL3 / Ras-related GTP-binding protein A / GATOR1 complex protein NPRL2 / Ras-related GTP-binding protein C
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsShen, K. / Huang, R.K. / Brignole, E.J. / Yu, Z. / 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 Institute Of Allergy and Infectious Diseases (NIH/NIAID)R37 AI47389 United States
Department of Defense (DOD, United States)W81XWH-15-1-0230 United States
National Science Foundation (NSF, United States)2016197106 United States
Howard Hughes Medical Institute (HHMI) United States
Life Sciences Research Foundation United States
CitationJournal: Nature / Year: 2018
Title: Architecture of the human GATOR1 and GATOR1-Rag GTPases complexes.
Authors: Kuang Shen / Rick K Huang / Edward J Brignole / Kendall J Condon / Max L Valenstein / Lynne Chantranupong / Aimaiti Bomaliyamu / Abigail Choe / Chuan Hong / Zhiheng Yu / David M Sabatini /
Abstract: Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase- ...Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.
History
DepositionFeb 12, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 28, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 11, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Apr 18, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Feb 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Apr 10, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.7Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.8Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
N: GATOR complex protein NPRL2
M: GATOR complex protein NPRL3
D: GATOR complex protein DEPDC5
A: Ras-related GTP-binding protein A
C: Ras-related GTP-binding protein C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)370,3066
Polymers369,7845
Non-polymers5221
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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GATOR complex protein ... , 3 types, 3 molecules NMD

#1: Protein GATOR complex protein NPRL2 / Gene 21 protein / G21 protein / Nitrogen permease regulator 2-like protein / NPR2-like protein / ...Gene 21 protein / G21 protein / Nitrogen permease regulator 2-like protein / NPR2-like protein / Tumor suppressor candidate 4


Mass: 43711.395 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NPRL2, TUSC4 / Cell line (production host): 293F / Production host: Homo sapiens (human) / References: UniProt: Q8WTW4
#2: Protein GATOR complex protein NPRL3 / -14 gene protein / Alpha-globin regulatory element-containing gene protein / Nitrogen permease ...-14 gene protein / Alpha-globin regulatory element-containing gene protein / Nitrogen permease regulator 3-like protein / Protein CGTHBA


Mass: 63680.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NPRL3, C16orf35, CGTHBA, MARE / Cell line (production host): 293F / Production host: Homo sapiens (human) / References: UniProt: Q12980
#3: Protein GATOR complex protein DEPDC5 / DEP domain-containing protein 5


Mass: 181478.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DEPDC5, KIAA0645 / Cell line (production host): 293F / Production host: Homo sapiens (human) / References: UniProt: O75140

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

#4: 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 / Plasmid: BL21(DE3) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q7L523
#5: Protein Ras-related GTP-binding protein C / RagC / GTPase-interacting protein 2 / TIB929


Mass: 44298.859 Da / Num. of mol.: 1 / Mutation: S75N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGC / Plasmid: BL21(DE3) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9HB90

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Non-polymers , 1 types, 1 molecules

#6: 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

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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: GATOR1-RAG / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.37 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 55 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128533 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00818857
ELECTRON MICROSCOPYf_angle_d1.40725533
ELECTRON MICROSCOPYf_dihedral_angle_d11.49311364
ELECTRON MICROSCOPYf_chiral_restr0.0712870
ELECTRON MICROSCOPYf_plane_restr0.013263

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