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- PDB-5h64: Cryo-EM structure of mTORC1 -

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Basic information

Entry
Database: PDB / ID: 5h64
TitleCryo-EM structure of mTORC1
Components
  • Regulatory-associated protein of mTOR
  • Serine/threonine-protein kinase mTOR
  • Target of rapamycin complex subunit LST8MTOR
KeywordsTRANSFERASE / cryo structure mTOR complex
Function / homology
Function and homology information


RNA polymerase III type 2 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of pentose-phosphate shunt / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / cellular response to leucine starvation / TFIIIC-class transcription factor complex binding / TORC2 complex ...RNA polymerase III type 2 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of pentose-phosphate shunt / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / cellular response to leucine starvation / TFIIIC-class transcription factor complex binding / TORC2 complex / heart valve morphogenesis / regulation of membrane permeability / negative regulation of lysosome organization / RNA polymerase III type 3 promoter sequence-specific DNA binding / TORC1 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / calcineurin-NFAT signaling cascade / regulation of autophagosome assembly / TORC1 signaling / positive regulation of odontoblast differentiation / voluntary musculoskeletal movement / regulation of osteoclast differentiation / positive regulation of keratinocyte migration / cellular response to L-leucine / MTOR signalling / Amino acids regulate mTORC1 / cellular response to nutrient / energy reserve metabolic process / Energy dependent regulation of mTOR by LKB1-AMPK / nucleus localization / ruffle organization / protein serine/threonine kinase inhibitor activity / negative regulation of cell size / cellular response to osmotic stress / positive regulation of osteoclast differentiation / enzyme-substrate adaptor activity / anoikis / cardiac muscle cell development / positive regulation of transcription by RNA polymerase III / negative regulation of protein localization to nucleus / regulation of myelination / negative regulation of calcineurin-NFAT signaling cascade / Macroautophagy / regulation of cell size / negative regulation of macroautophagy / lysosome organization / positive regulation of oligodendrocyte differentiation / positive regulation of actin filament polymerization / protein kinase activator activity / positive regulation of myotube differentiation / behavioral response to pain / TOR signaling / oligodendrocyte differentiation / mTORC1-mediated signalling / germ cell development / Constitutive Signaling by AKT1 E17K in Cancer / social behavior / cellular response to nutrient levels / CD28 dependent PI3K/Akt signaling / positive regulation of phosphoprotein phosphatase activity / positive regulation of translational initiation / neuronal action potential / HSF1-dependent transactivation / positive regulation of TOR signaling / positive regulation of G1/S transition of mitotic cell cycle / positive regulation of epithelial to mesenchymal transition / regulation of macroautophagy / endomembrane system / 'de novo' pyrimidine nucleobase biosynthetic process / response to amino acid / positive regulation of lipid biosynthetic process / phagocytic vesicle / positive regulation of lamellipodium assembly / heart morphogenesis / regulation of cellular response to heat / cytoskeleton organization / cardiac muscle contraction / positive regulation of stress fiber assembly / cellular response to amino acid starvation / T cell costimulation / cellular response to starvation / positive regulation of endothelial cell proliferation / protein serine/threonine kinase activator activity / positive regulation of glycolytic process / negative regulation of autophagy / response to nutrient levels / post-embryonic development / response to nutrient / 14-3-3 protein binding / positive regulation of translation / VEGFR2 mediated vascular permeability / regulation of signal transduction by p53 class mediator / positive regulation of peptidyl-threonine phosphorylation / Regulation of PTEN gene transcription / regulation of autophagy / regulation of cell growth / regulation of actin cytoskeleton organization / cellular response to glucose stimulus / cellular response to amino acid stimulus / TP53 Regulates Metabolic Genes
Similarity search - Function
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 / Domain of unknown function DUF3385, target of rapamycin protein / Domain of unknown function (DUF3385) / Domain of unknown function / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR ...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 / Domain of unknown function DUF3385, target of rapamycin protein / Domain of unknown function (DUF3385) / Domain of unknown function / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR / FKBP12-rapamycin binding domain superfamily / FKBP12-rapamycin binding domain / HEAT repeat / HEAT repeat / Rapamycin binding domain / PIK-related kinase, FAT / FAT domain / FATC 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 / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Serine/threonine-protein kinase mTOR / Regulatory-associated protein of mTOR / Target of rapamycin complex subunit LST8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsYang, H. / Wang, J. / Liu, M. / Chen, X. / Huang, M. / Tan, D. / Dong, M. / Wong, C.C.L. / Wang, J. / Xu, Y. / Wang, H.
Funding support China, 5items
OrganizationGrant numberCountry
National Natural Science Foundation of ChinaU1432242 China
National Natural Science Foundation of China31425008 China
National Natural Science Foundation of China91419301 China
Basic Research Project of Shanghai Science and Technology Commission12JC1402700 China
Program of Shanghai Subject Chief Scientist14XD1400500 China
CitationJournal: Protein Cell / Year: 2016
Title: 4.4 Å Resolution Cryo-EM structure of human mTOR Complex 1.
Authors: Huirong Yang / Jia Wang / Mengjie Liu / Xizi Chen / Min Huang / Dan Tan / Meng-Qiu Dong / Catherine C L Wong / Jiawei Wang / Yanhui Xu / Hong-Wei Wang /
Abstract: Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through ...Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through regulating translation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 Å resolution. The mTORC1 comprises a dimer of heterotrimer (mTOR-Raptor-mLST8) mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspase-like domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for substrate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance for substrate entry to the catalytic cavity of mTORC1. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regulatory mechanism of mTORC1 pathway.
History
DepositionNov 10, 2016Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 25, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2017Group: Database references
Revision 1.2Dec 6, 2017Group: Advisory / Data processing / Database references
Category: citation / citation_author ...citation / citation_author / em_software / pdbx_unobs_or_zero_occ_atoms
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _em_software.name
Revision 1.3Nov 6, 2019Group: Data collection / Other / Category: atom_sites / cell
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

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Structure visualization

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  • Deposited structure unit
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  • EMDB-6668
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Assembly

Deposited unit
A: Serine/threonine-protein kinase mTOR
B: Regulatory-associated protein of mTOR
C: Target of rapamycin complex subunit LST8
a: Serine/threonine-protein kinase mTOR
b: Regulatory-associated protein of mTOR
c: Target of rapamycin complex subunit LST8


Theoretical massNumber of molelcules
Total (without water)948,7366
Polymers948,7366
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10360 Å2
ΔGint-60 kcal/mol
Surface area299360 Å2

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Components

#1: Protein 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: 289257.969 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 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
References: UniProt: P42345, non-specific serine/threonine protein kinase
#2: 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 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q8N122
#3: Protein Target of rapamycin complex subunit LST8 / MTOR / 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 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q9BVC4

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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: human mTOR complex 1MTORC1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mammalia (mammals) / Plasmid: pCAG
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMNaClSodium chloride1
31 mMTCEP1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 22500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 8.25 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2997
Image scansWidth: 7676 / Height: 7420 / Movie frames/image: 32 / Used frames/image: 0-31

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
2UCSFImage4image acquisition
4CTFFIND33CTF correction
7EMfitmodel fitting
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
12RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 486584
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115039 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01149020
ELECTRON MICROSCOPYf_angle_d1.46666566
ELECTRON MICROSCOPYf_dihedral_angle_d8.46839286
ELECTRON MICROSCOPYf_chiral_restr0.0647738
ELECTRON MICROSCOPYf_plane_restr0.0088636

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