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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/homologyRapamycin-insensitive companion of mTOR, phosphorylation-site / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Pianissimo family / Rapamycin-insensitive companion of mTOR, N-terminal domain / 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, middle domain / Rapamycin-insensitive companion of mTOR, N-term ...Rapamycin-insensitive companion of mTOR, phosphorylation-site / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Pianissimo family / Rapamycin-insensitive companion of mTOR, N-terminal domain / 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, middle domain / Rapamycin-insensitive companion of mTOR, N-term / Rapamycin-insensitive companion of mTOR, domain 5 / Stress-activated map kinase interacting protein 1 (SIN1) / Sin1, N-terminal / Sin1, middle CRIM domain / SAPK-interacting protein 1, Pleckstrin-homology domain / TORC2 component Sin1/Avo1 / SAPK-interacting protein 1 (Sin1), middle CRIM domain / SAPK-interacting protein 1 (Sin1), Pleckstrin-homology / TORC2 signaling / regulation of protein kinase B signaling / Target of rapamycin complex subunit LST8 / regulation of peptidyl-serine phosphorylation / establishment or maintenance of actin cytoskeleton polarity / negative regulation of iodide transmembrane transport / positive regulation of skeletal muscle hypertrophy / positive regulation of wound healing, spreading of epidermal cells / negative regulation of cholangiocyte apoptotic process / positive regulation of granulosa cell proliferation / positive regulation of cholangiocyte proliferation / negative regulation of muscle atrophy / RNA polymerase III type 2 promoter DNA binding / RNA polymerase III type 1 promoter DNA binding / regulation of fatty acid beta-oxidation / positive regulation of eating behavior / Domain of unknown function DUF3385, target of rapamycin protein / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR / FKBP12-rapamycin binding domain superfamily / RNA polymerase III type 3 promoter DNA binding / cellular response to leucine starvation / regulation of carbohydrate utilization / cardiac muscle cell development / Domain of unknown function (DUF3385) / Ras GTPase binding / regulation of response to food / TFIIIC-class transcription factor binding / cellular response to leucine / TORC1 complex / heart valve morphogenesis / TORC1 signaling / positive regulation of sensory perception of pain / phosphatidic acid binding / FKBP12-rapamycin binding domain / regulation of membrane permeability / positive regulation of glial cell proliferation / positive regulation of keratinocyte migration / regulation of brown fat cell differentiation / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / phosphatidylinositol-3,5-bisphosphate binding / PIK-related kinase, FAT / TORC2 complex / regulation of glycogen biosynthetic process / voluntary musculoskeletal movement / FATC domain profile. / PIK-related kinase / FAT domain profile. / positive regulation of neuron maturation / FATC domain / energy reserve metabolic process / mTOR signalling / regulation of osteoclast differentiation / FAT domain / regulation of phosphorylation / phosphatidylinositol-3,4-bisphosphate binding / FATC domain / cellular response to nutrient levels / mRNA stabilization / Energy dependent regulation of mTOR by LKB1-AMPK / positive regulation of TOR signaling / negative regulation of cell size / go:0051534: / embryo development / regulation of establishment of cell polarity / ruffle organization / negative regulation of Ras protein signal transduction / anoikis / mTORC1-mediated signalling / phosphatidylinositol 3-kinase complex / establishment of cell polarity / positive regulation of transcription by RNA polymerase III / regulation of myelination / positive regulation of cell growth involved in cardiac muscle cell development / positive regulation of lamellipodium assembly / Quinoprotein alcohol dehydrogenase-like superfamily / regulation of cell size / spinal cord development / negative regulation of macroautophagy / TOR signaling / positive regulation of dendritic spine development / Macroautophagy
Function and homology information
Specimen sourceHomo sapiens / human /
MethodElectron microscopy (4.9 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsChen, X. / Liu, M. / Tian, Y. / Wang, H. / Wang, J. / Xu, Y.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 20, 2018 / Release: Mar 21, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 21, 2018Structure modelrepositoryInitial release
1.1Apr 4, 2018Structure modelData collection / Database referencescitation / citation_author_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

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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
Polyers1,110,9388
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide Serine/threonine-protein kinase mTOR / 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: (gene. exp.) Homo sapiens / human / / Gene: MTOR, FRAP, FRAP1, FRAP2, RAFT1, RAPT1 / Cell line (production host): 293F / Production host: Homo sapiens
References: UniProt:P42345, EC:2.7.11.1 (non-specific serine/threonine protein kinase)
#2: Protein/peptide Target of rapamycin complex subunit LST8 / 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: (gene. exp.) Homo sapiens / human / / Gene: MLST8, GBL, LST8 / Cell line (production host): 293F / Production host: Homo sapiens / References: UniProt:Q9BVC4
#3: Protein/peptide Rapamycin-insensitive companion of mTOR / AVO3 homolog / hAVO3


Mass: 175142.500 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens / human / / Gene: RICTOR, KIAA1999 / Cell line (production host): 293F / Production host: Homo sapiens / References: UniProt:Q6R327
#4: Protein/peptide Target of rapamycin complex 2 subunit MAPKAP1 / 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: (gene. exp.) Homo sapiens / human / / Gene: MAPKAP1, MIP1, SIN1 / Cell line (production host): 293F / Production host: Homo sapiens / 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 / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: human mTOR Complex 2 / Type: COMPLEX / Entity ID: 1, 2, 3, 4 / Source: RECOMBINANT
Molecular weightValue: 1400 deg. / Units: KILODALTONS/NANOMETER / Experimental value: YES
Source (natural)Organism: Homo sapiens
Source (recombinant)Cell: 293F / Organism: Homo sapiens
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 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 282 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: 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 mm
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
10RELION2.0FINAL EULER ASSIGNMENT
12RELION2.0RECONSTRUCTION
13PHENIXMODEL REFINEMENT
CTF correctionType: NONE
SymmetryPoint symmetry: C2
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 195353 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref 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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