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- PDB-6sb2: cryo-EM structure of mTORC1 bound to active RagA/C GTPases -

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Basic information

Entry
Database: PDB / ID: 6sb2
Titlecryo-EM structure of mTORC1 bound to active RagA/C GTPases
Components
  • (Ras-related GTP-binding protein ...) x 2
  • Regulatory-associated protein of mTOR
  • Target of rapamycin complex subunit LST8
  • mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-protein kinase mTOR
KeywordsSIGNALING PROTEIN / small GTPases / mTORC1 activator / roadblock domain / GTPase domain
Function / homology
Function and homology information


Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / cardiac cell development / positive regulation of SCF-dependent proteasomal ubiquitin-dependent catabolic process / RNA polymerase III type 2 promoter sequence-specific DNA binding / T-helper 1 cell lineage commitment / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / regulation of locomotor rhythm / positive regulation of pentose-phosphate shunt ...Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / cardiac cell development / positive regulation of SCF-dependent proteasomal ubiquitin-dependent catabolic process / RNA polymerase III type 2 promoter sequence-specific DNA binding / T-helper 1 cell lineage commitment / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / regulation of locomotor rhythm / positive regulation of pentose-phosphate shunt / positive regulation of wound healing, spreading of epidermal cells / regulation of TORC1 signaling / TORC2 complex / cellular response to leucine starvation / TFIIIC-class transcription factor complex binding / positive regulation of odontoblast differentiation / regulation of lysosome organization / protein localization to lysosome / TORC1 complex / negative regulation of lysosome organization / regulation of osteoclast differentiation / RNA polymerase III type 3 promoter sequence-specific DNA binding / positive regulation of keratinocyte migration / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / MTOR signalling / cellular response to L-leucine / Energy dependent regulation of mTOR by LKB1-AMPK / cellular response to nutrient / regulation of autophagosome assembly / Amino acids regulate mTORC1 / Dengue virus modulates apoptosis / cellular response to methionine / positive regulation of osteoclast differentiation / TORC2 signaling / cellular response to osmotic stress / TORC1 signaling / anoikis / protein localization to membrane / inositol hexakisphosphate binding / negative regulation of protein localization to nucleus / positive regulation of ubiquitin-dependent protein catabolic process / regulation of cell size / regulation of TOR signaling / negative regulation of macroautophagy / Macroautophagy / small GTPase-mediated signal transduction / Constitutive Signaling by AKT1 E17K in Cancer / behavioral response to pain / positive regulation of transcription by RNA polymerase III / positive regulation of protein kinase activity / social behavior / response to amino acid / neuronal action potential / TOR signaling / mTORC1-mediated signalling / CD28 dependent PI3K/Akt signaling / HSF1-dependent transactivation / positive regulation of TOR signaling / positive regulation of translational initiation / positive regulation of lipid biosynthetic process / protein serine/threonine kinase inhibitor activity / enzyme-substrate adaptor activity / protein kinase activator activity / positive regulation of epithelial to mesenchymal transition / T cell costimulation / vascular endothelial cell response to laminar fluid shear stress / positive regulation of G1/S transition of mitotic cell cycle / regulation of cellular response to heat / cellular response to nutrient levels / regulation of macroautophagy / 'de novo' pyrimidine nucleobase biosynthetic process / cytoskeleton organization / positive regulation of endothelial cell proliferation / negative regulation of insulin receptor signaling pathway / phagocytic vesicle / protein-membrane adaptor activity / 14-3-3 protein binding / positive regulation of TORC1 signaling / endomembrane system / RNA splicing / positive regulation of glycolytic process / negative regulation of autophagy / protein serine/threonine kinase activator activity / cellular response to amino acid stimulus / regulation of signal transduction by p53 class mediator / cellular response to amino acid starvation / cellular response to starvation / Regulation of PTEN gene transcription / tumor necrosis factor-mediated signaling pathway / phosphatidylinositol 3-kinase/protein kinase B signal transduction / VEGFR2 mediated vascular permeability / regulation of actin cytoskeleton organization / positive regulation of translation / TP53 Regulates Metabolic Genes / non-specific protein-tyrosine kinase / cellular response to glucose stimulus / regulation of cell growth / non-membrane spanning protein tyrosine kinase activity / phosphoprotein binding / response to nutrient levels
Similarity search - Function
RagA/B / RagC/D / Gtr1/RagA G protein / Gtr1/RagA G protein conserved region / Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / Target of rapamycin complex subunit LST8 / HEAT repeat ...RagA/B / RagC/D / Gtr1/RagA G protein / Gtr1/RagA G protein conserved region / Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / Target of rapamycin complex subunit LST8 / HEAT repeat / HEAT repeat / Domain of unknown function DUF3385, target of rapamycin protein / Serine/threonine-protein kinase mTOR domain / Domain of unknown function / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR / FKBP12-rapamycin binding domain superfamily / FKBP12-rapamycin binding domain / Rapamycin binding domain / : / Serine/threonine-protein kinase ATR-like, HEAT repeats / : / FATC domain / PIK-related kinase, FAT / FAT domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Quinoprotein alcohol dehydrogenase-like superfamily / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / WD domain, G-beta repeat / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Serine/threonine-protein kinase mTOR / Ras-related GTP-binding protein A / Regulatory-associated protein of mTOR / Target of rapamycin complex subunit LST8 / Ras-related GTP-binding protein C
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsAnandapadamanaban, M. / Berndt, A. / Masson, G.R. / Perisic, O. / Williams, R.L.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184308 United Kingdom
Cancer Research UKC14801/A21211 United Kingdom
European Molecular Biology Organizationlong-term fellowship United Kingdom
CitationJournal: Science / Year: 2019
Title: Architecture of human Rag GTPase heterodimers and their complex with mTORC1.
Authors: Madhanagopal Anandapadamanaban / Glenn R Masson / Olga Perisic / Alex Berndt / Jonathan Kaufman / Chris M Johnson / Balaji Santhanam / Kacper B Rogala / David M Sabatini / Roger L Williams /
Abstract: The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag ...The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagA/RagC nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
History
DepositionJul 18, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 22, 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: mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-protein kinase mTOR
B: mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-protein kinase mTOR
E: Target of rapamycin complex subunit LST8
C: Ras-related GTP-binding protein A
D: Ras-related GTP-binding protein C
Y: Regulatory-associated protein of mTOR
H: Target of rapamycin complex subunit LST8
I: Ras-related GTP-binding protein A
J: Ras-related GTP-binding protein C
N: Regulatory-associated protein of mTOR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,108,39314
Polymers1,106,46110
Non-polymers1,9334
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, HDX-MS and SEC-MALLS provides evidence of mTORC1-RagA/C dimer in solution.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 6 molecules ABEHYN

#1: Protein mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-protein kinase mTOR / FK506-binding protein 12-rapamycin complex-associated protein 1 / FKBP12-rapamycin complex- ...FK506-binding protein 12-rapamycin complex-associated protein 1 / FKBP12-rapamycin complex-associated protein / Mammalian target of rapamycin / mTOR / Mechanistic target of rapamycin / Rapamycin and FKBP12 target 1 / Rapamycin target protein 1


Mass: 287235.188 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MTOR, FRAP, FRAP1, FRAP2, RAFT1, RAPT1 / Production host: Homo sapiens (human)
References: UniProt: P42345, non-specific serine/threonine protein kinase
#2: Protein Target of rapamycin complex subunit LST8 / TORC subunit LST8 / G protein beta subunit-like / Protein GbetaL / Mammalian lethal with SEC13 ...TORC subunit LST8 / G protein beta subunit-like / Protein GbetaL / Mammalian lethal with SEC13 protein 8 / mLST8


Mass: 35910.090 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MLST8, GBL, LST8 / Production host: Homo sapiens (human) / References: UniProt: Q9BVC4
#5: Protein Regulatory-associated protein of mTOR / Raptor / p150 target of rapamycin (TOR)-scaffold protein


Mass: 149200.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPTOR, KIAA1303, RAPTOR / Production host: Escherichia coli (E. coli) / References: UniProt: Q8N122

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

#3: Protein Ras-related GTP-binding protein 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: Escherichia coli (E. coli) / References: UniProt: Q7L523
#4: Protein Ras-related GTP-binding protein C / RagC / GTPase-interacting protein 2 / TIB929


Mass: 44284.832 Da / Num. of mol.: 2
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 , 2 types, 4 molecules

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


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#7: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


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

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Details

Has ligand of interestY

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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
IDNameTypeEntity IDParent-IDSource
1cryo-EM structure of mTORC1 bound to active RagA/C complexCOMPLEX#1-#50MULTIPLE SOURCES
2mTORC1COMPLEX#1-#21RECOMBINANT
3RagA/CCOMPLEX#3-#51RECOMBINANT
Molecular weightValue: 1.09 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli (E. coli)562
Buffer solutionpH: 7
Details: 100mM Tris-HCl pH7.0, 260mM NaCl, 5mM MgCl2, 1mM TCEP
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: mTORC1 (mTOR complex 1) is a dimer consists of three proteins: mTOR, mLST8 and RAPTOR. The small GTPases, RagA/C in its active form bind to mTORC1 for activation. We solved the cryo-EM ...Details: mTORC1 (mTOR complex 1) is a dimer consists of three proteins: mTOR, mLST8 and RAPTOR. The small GTPases, RagA/C in its active form bind to mTORC1 for activation. We solved the cryo-EM structure of mTORC1 bound to RagA/C.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 %

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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 / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.8 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 22 / Used frames/image: 1-22

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Processing

EM software
IDNameVersionCategory
1RELION3.0.6particle selection
2EPU1.10.0.77RELimage acquisition
4Gctf1.18CTF correction
7REFMAC5.8.0238model fitting
9RELION3.0.6initial Euler assignment
10RELION3.0.6final Euler assignment
11RELION3.0.6classification
12RELION3.0.63D reconstruction
13REFMAC5.8.0238model refinement
Image processingDetails: The selected images were processed using MotionCor2 within the RELION-3.0.6 package.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 169971
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51902 / Algorithm: FOURIER SPACE
Details: For the final reconstruction of mTORC1-RagA/C structure we used a strategy taking advantage of the relion particle symmetry expand program, and duplicated the C2-refined particles and ...Details: For the final reconstruction of mTORC1-RagA/C structure we used a strategy taking advantage of the relion particle symmetry expand program, and duplicated the C2-refined particles and applied the appropriate rotation and translation to generate a set of monomers. We performed mTORC1-RagA/C 'pseudo-monomer' focussed classification with signal subtraction and obtained a reconstruction of 6.2 A resolution map. This cryo-EM density corresponded to the mTORC1-RagA/C pseudomonomer, where the previously published structure for apo-mTORC1 (PDB ID 6BCX) and our high-resolution crystal structure of RagA/C (6S6A) were fitted with great confidence from our experimental analysis including Pulldown assays, mutational at per-residue level in the binding interface and HDX-Mass Spectrometry.
Symmetry type: POINT
Atomic model buildingB value: 315 / Protocol: RIGID BODY FIT / Space: REAL
Details: Cryo-EM model of mTORC1-RagA/C was refined using REFMAC5 program in CCPEM package, with a composite map of the 3D reconstruction of mTORC1-RagA/C pseudo-monomer (as mentioned in ...Details: Cryo-EM model of mTORC1-RagA/C was refined using REFMAC5 program in CCPEM package, with a composite map of the 3D reconstruction of mTORC1-RagA/C pseudo-monomer (as mentioned in Reconstruction section) of one protomer together with the generated map for the other second protomer of mTORC1-RagA/C. This second protomer of mTORC1-RagA/C map was generated by simply aligning the first 3D reconstructed pseudomonomer map onto the mTORC1 dimer consensus C2 map and then obtained the rotation-translation matrix with CHIMERA and then used Maputils program in CCP4i. From the resulting mTORC1-RagA/C dimer map, the model of mTORC1-RagA/C was built by using previously published structure of apo-mTORC1 (PDB ID 6BCX) and our crystal structure of RagA/C was fitted (PDB ID 6S6A, unreleased). The entire mTORC1-RagA/C final model was refined using REFMAC5 program using the restraints from the crystal structure of RagA/C and previously published mTORC1 structure. Side chains were removed before refinement, since these were not evident in the cryo-EM densities. Separate model refinements were performed against single half-maps, and the resulting models were compared with the other half-maps to confirm the absence of overfitting.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16BCX

6bcx

16BCX1PDBexperimental model
26S6A

6s6a

16S6A2PDBexperimental model

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