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- PDB-5zcs: 4.9 Angstrom Cryo-EM structure of human mTOR complex 2 -

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Entry
Database: PDB / ID: 5zcs
Title4.9 Angstrom Cryo-EM structure of human mTOR complex 2
Components
  • Rapamycin-insensitive companion of mTOR
  • Serine/threonine-protein kinase mTOR
  • Target of rapamycin complex 2 subunit MAPKAP1
  • Target of rapamycin complex subunit LST8
KeywordsGENE REGULATION / Cryo-EM structure human mTORC2
Function / homology
Function and homology information


TORC2 signaling / regulation of peptidyl-serine phosphorylation / RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / TORC2 complex ...TORC2 signaling / regulation of peptidyl-serine phosphorylation / RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / TORC2 complex / cellular response to leucine starvation / TFIIIC-class transcription factor complex binding / regulation of membrane permeability / heart valve morphogenesis / negative regulation of lysosome organization / nucleus localization / RNA polymerase III type 3 promoter sequence-specific DNA binding / TORC1 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / regulation of cellular response to oxidative stress / calcineurin-NFAT signaling cascade / regulation of osteoclast differentiation / voluntary musculoskeletal movement / TORC1 signaling / positive regulation of keratinocyte migration / phosphatidic acid binding / cellular response to L-leucine / Amino acids regulate mTORC1 / MTOR signalling / cellular response to nutrient / cellular response to methionine / Energy dependent regulation of mTOR by LKB1-AMPK / regulation of autophagosome assembly / energy reserve metabolic process / negative regulation of cell size / ruffle organization / negative regulation of Ras protein signal transduction / phosphatidylinositol-3,4-bisphosphate binding / cellular response to osmotic stress / phosphatidylinositol-3,5-bisphosphate binding / anoikis / cardiac muscle cell development / negative regulation of protein localization to nucleus / embryo development ending in birth or egg hatching / regulation of establishment of cell polarity / positive regulation of transcription by RNA polymerase III / negative regulation of calcineurin-NFAT signaling cascade / regulation of myelination / positive regulation of actin filament polymerization / regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / regulation of cell size / negative regulation of macroautophagy / positive regulation of oligodendrocyte differentiation / lysosome organization / Macroautophagy / positive regulation of myotube differentiation / behavioral response to pain / oligodendrocyte differentiation / Constitutive Signaling by AKT1 E17K in Cancer / mTORC1-mediated signalling / germ cell development / phosphatidylinositol-3,4,5-trisphosphate binding / CD28 dependent PI3K/Akt signaling / : / HSF1-dependent transactivation / positive regulation of TOR signaling / neuronal action potential / response to amino acid / TOR signaling / 'de novo' pyrimidine nucleobase biosynthetic process / endomembrane system / regulation of macroautophagy / positive regulation of translational initiation / cellular response to nutrient levels / positive regulation of lamellipodium assembly / phosphorylation / phagocytic vesicle / positive regulation of lipid biosynthetic process / heart morphogenesis / positive regulation of epithelial to mesenchymal transition / cardiac muscle contraction / regulation of cellular response to heat / phosphatidylinositol-4,5-bisphosphate binding / positive regulation of stress fiber assembly / cytoskeleton organization / positive regulation of endothelial cell proliferation / T cell costimulation / substantia nigra development / cellular response to starvation / cellular response to amino acid starvation / post-embryonic development / positive regulation of glycolytic process / protein serine/threonine kinase activator activity / negative regulation of autophagy / response to nutrient / response to nutrient levels / VEGFR2 mediated vascular permeability / regulation of signal transduction by p53 class mediator / Regulation of PTEN gene transcription / positive regulation of translation
Similarity search - Function
Rapamycin-insensitive companion of mTOR, N-terminal domain / Pianissimo family / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Rapamycin-insensitive companion of mTOR, middle domain / Rapamycin-insensitive companion of mTOR, domain 5 / Rapamycin-insensitive companion of mTOR, domain 4 / Rapamycin-insensitive companion of mTOR RasGEF_N domain / Rapamycin-insensitive companion of mTOR, N-term / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Rapamycin-insensitive companion of mTOR, middle domain ...Rapamycin-insensitive companion of mTOR, N-terminal domain / Pianissimo family / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Rapamycin-insensitive companion of mTOR, middle domain / Rapamycin-insensitive companion of mTOR, domain 5 / Rapamycin-insensitive companion of mTOR, domain 4 / Rapamycin-insensitive companion of mTOR RasGEF_N domain / Rapamycin-insensitive companion of mTOR, N-term / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Rapamycin-insensitive companion of mTOR, middle domain / Rapamycin-insensitive companion of mTOR, domain 5 / Rapamycin-insensitive companion of mTOR RasGEF_N domain / Rapamycin-insensitive companion of mTOR, middle domain / Rapamycin-insensitive companion of mTOR, N-term / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Rapamycin-insensitive companion of mTOR, domain 5 / Sin1, N-terminal / Stress-activated map kinase interacting protein 1 (SIN1) / TORC2 component Sin1/Avo1 / SAPK-interacting protein 1, Pleckstrin-homology domain / Sin1, middle CRIM domain / SAPK-interacting protein 1 (Sin1), middle CRIM domain / SAPK-interacting protein 1 (Sin1), Pleckstrin-homology / Target of rapamycin complex subunit LST8 / 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 / : / PIK-related kinase, FAT / FATC domain / FATC / FAT domain / FAT domain profile. / FATC domain profile. / FATC domain / PIK-related kinase / Quinoprotein alcohol dehydrogenase-like superfamily / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / PH-like domain superfamily / 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. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Serine/threonine-protein kinase mTOR / Rapamycin-insensitive companion of mTOR / Target of rapamycin complex 2 subunit MAPKAP1 / Target of rapamycin complex subunit LST8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsChen, X. / Liu, M. / Tian, Y. / Wang, H. / Wang, J. / Xu, Y.
Funding support China, 4items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2016YFA0500700,2016YFA0501100 China
National Natural Science Foundation of China31770781,U1432242,31425008,91419301 China
Strategic Priority Research Program of the Chinese Academy of SciencesXDB08000000 China
National Program for support of Top-Notch Young Professionals China
CitationJournal: Cell Res / Year: 2018
Title: Cryo-EM structure of human mTOR complex 2.
Authors: Xizi Chen / Mengjie Liu / Yuan Tian / Jiabei Li / Yilun Qi / Dan Zhao / Zihan Wu / Min Huang / Catherine C L Wong / Hong-Wei Wang / Jiawei Wang / Huirong Yang / Yanhui Xu /
Abstract: Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and ...Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and SGK1. The functional core complex consists of mTOR, mLST8, and two mTORC2-specific components, Rictor and mSin1. Here we investigated the intermolecular interactions within mTORC2 complex and determined its cryo-electron microscopy structure at 4.9 Å resolution. The structure reveals a hollow rhombohedral fold with a 2-fold symmetry. The dimerized mTOR serves as a scaffold for the complex assembly. The N-terminal half of Rictor is composed of helical repeat clusters and binds to mTOR through multiple contacts. mSin1 is located close to the FRB domain and catalytic cavity of mTOR. Rictor and mSin1 together generate steric hindrance to inhibit binding of FKBP12-rapamycin to mTOR, revealing the mechanism for rapamycin insensitivity of mTORC2. The mTOR dimer in mTORC2 shows more compact conformation than that of mTORC1 (rapamycin sensitive), which might result from the interaction between mTOR and Rictor-mSin1. Structural comparison shows that binding of Rictor and Raptor (mTORC1-specific component) to mTOR is mutually exclusive. Our study provides a basis for understanding the assembly of mTORC2 and a framework to further characterize the regulatory mechanism of mTORC2 pathway.
History
DepositionFeb 20, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 21, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 4, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 30, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
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
Revision 1.4Mar 27, 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
A: Serine/threonine-protein kinase mTOR
B: Serine/threonine-protein kinase mTOR
C: Target of rapamycin complex subunit LST8
D: Target of rapamycin complex subunit LST8
E: Rapamycin-insensitive companion of mTOR
F: Rapamycin-insensitive companion of mTOR
G: Target of rapamycin complex 2 subunit MAPKAP1
H: Target of rapamycin complex 2 subunit MAPKAP1


Theoretical massNumber of molelcules
Total (without water)1,110,9388
Polymers1,110,9388
Non-polymers00
Water00
1


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

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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): 293F / 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 / Cell line (production host): 293F / Production host: Homo sapiens (human) / References: UniProt: Q9BVC4
#3: Protein Rapamycin-insensitive companion of mTOR / AVO3 homolog / hAVO3


Mass: 175142.500 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RICTOR, KIAA1999 / Cell line (production host): 293F / Production host: Homo sapiens (human) / References: UniProt: Q6R327
#4: Protein Target of rapamycin complex 2 subunit MAPKAP1 / SIN:Stress-activated map kinase-interacting protein 1 / TORC2 subunit MAPKAP1 / Mitogen-activated ...SIN:Stress-activated map kinase-interacting protein 1 / TORC2 subunit MAPKAP1 / Mitogen-activated protein kinase 2-associated protein 1 / Stress-activated map kinase-interacting protein 1 / mSIN1


Mass: 55158.477 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAPKAP1, MIP1, SIN1 / Cell line (production host): 293F / Production host: Homo sapiens (human) / References: UniProt: Q9BPZ7
Sequence details1. FOR ENTITY 3 (CHAINS E/F), ENTIRE SEQUENCE HAS BEEN USED IN THE EXPERIMENT. HOWEVER, C-TERMINAL ...1. FOR ENTITY 3 (CHAINS E/F), ENTIRE SEQUENCE HAS BEEN USED IN THE EXPERIMENT. HOWEVER, C-TERMINAL THREE HELIX CANNOT BE ASSIGNED ACCURATELY, AND WERE ASSIGNED TO RESIDUE NUMBER 1610-1626, 1630-1649 AND 1680-1695 TEMPORARY. THE REAL SEQUENCE FOR RESIDUE 1019-1708 SHOULD BE PSTLSLNSESTSSRHNSESESVPSSMFILEDDRFGSSSTSTFFLDINEDTEPTFYDRSGPIKDKNSFPFFASSKLVKNRILNSLTLPNKKHRSSSDPKGGKLSSESKTSNRRIRTLTEPSVDFNHSDDFTPISTVQKTLQLETS FMGNKHIEDTGSTPSIGENDLKFTKNFGTENHRENTSRERLVVESSTSSHMKIRSQSFNTDTTTSGISSMSSSPSRETVGVDATTMDTDCGSMSTVVSTKTIKTSHYLTPQSNHLSLSKSNSVSLVPPGSSHTLPRRAQSLKAP SIATIKSLADCNFSYTSSRDAFGYATLKRLQQQRMHPSLSHSEALASPAKDVLFTDTITMKANSFESRLTPSRFMKALSYASLDKEDLLSPINQNTLQRSSSVRSMVSSATYGGSDDYIGLALPVDINDIFQVKDIPYFQTKNI PPHDDRGARAFAHDAGGLPSGTGGLVKNSFHLLRQQMSLTEIMNSIHSDASLFLESTEDTGLQEHTDDNCLYCVCIEILGFQPSNQLSAICSHSDFQDIPYSDWCEQTIHNPLEVVPSKFSGISGCSDGVSQEGSASSTKSTEL LLGVKTIPDDTPMCRILLRKEVLRLVINLSSSVSTKCHETGLLTIKEKYPQTFDDICLYSEVSHLLSHCTFRLPCRRFIQELFQDVQFLQMHEEAEAVLATPPKQPIVDTSAES 2. FOR ENTITY 4 (CHAINS G/H), ENTIRE SEQUENCE HAS BEEN USED IN THE EXPERIMENT. HOWEVER, ONLY N-TERMINAL RESIDUES (SUPPOSED TO BE WITHIN RESIDUE RANGE 1-140) WERE ASSIGNED, BUT NOT ACCURATELY. THE REAL SEQUENCE FOR RESIDUE 1-140 SHOULD BE: MAFLDNPTIILAHIRQSHVTSDDTGMCEMVLIDHDVDLEKIHPPSMPGDSGSEIQGSNGETQGYVYAQSVDITSSWDFGIRRRSNTAQRLERLRKERQNQIKCKNIQWKERNSKQSAQELKSLFEKKSLKEKPPISGKQS

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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 2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1400 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: 293F
Buffer solutionpH: 7.4
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 282 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 32 / Used frames/image: 1-32

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.6image acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
10RELION2final Euler assignment
12RELION23D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 195353 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01350040
ELECTRON MICROSCOPYf_angle_d1.53268210
ELECTRON MICROSCOPYf_dihedral_angle_d7.70530230
ELECTRON MICROSCOPYf_chiral_restr0.0688168
ELECTRON MICROSCOPYf_plane_restr0.0098892

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