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- PDB-6zwm: cryo-EM structure of human mTOR complex 2, overall refinement -

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Basic information

Entry
Database: PDB / ID: 6zwm
Titlecryo-EM structure of human mTOR complex 2, overall refinement
Components
  • (Target of rapamycin complex ...) x 2
  • Rapamycin-insensitive companion of mTOR
  • Serine/threonine-protein kinase mTOR
KeywordsTRANSFERASE / mTOR / Kinase / Complex / Inositol
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
ACETYL GROUP / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / INOSITOL HEXAKISPHOSPHATE / 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: 3.2 Å
AuthorsScaiola, A. / Mangia, F. / Imseng, S. / Boehringer, D. / Ban, N. / Maier, T.
Funding support Switzerland, 3items
OrganizationGrant numberCountry
Swiss National Science Foundation179323 Switzerland
Swiss National Science Foundation177084 Switzerland
Swiss National Science Foundation138262
CitationJournal: Sci Adv / Year: 2020
Title: The 3.2-Å resolution structure of human mTORC2.
Authors: Alain Scaiola / Francesca Mangia / Stefan Imseng / Daniel Boehringer / Karolin Berneiser / Mitsugu Shimobayashi / Edward Stuttfeld / Michael N Hall / Nenad Ban / Timm Maier /
Abstract: The protein kinase mammalian target of rapamycin (mTOR) is the central regulator of cell growth. Aberrant mTOR signaling is linked to cancer, diabetes, and neurological disorders. mTOR exerts its ...The protein kinase mammalian target of rapamycin (mTOR) is the central regulator of cell growth. Aberrant mTOR signaling is linked to cancer, diabetes, and neurological disorders. mTOR exerts its functions in two distinct multiprotein complexes, mTORC1 and mTORC2. Here, we report a 3.2-Å resolution cryo-EM reconstruction of mTORC2. It reveals entangled folds of the defining Rictor and the substrate-binding SIN1 subunits, identifies the carboxyl-terminal domain of Rictor as the source of the rapamycin insensitivity of mTORC2, and resolves mechanisms for mTORC2 regulation by complex destabilization. Two previously uncharacterized small-molecule binding sites are visualized, an inositol hexakisphosphate (InsP6) pocket in mTOR and an mTORC2-specific nucleotide binding site in Rictor, which also forms a zinc finger. Structural and biochemical analyses suggest that InsP6 and nucleotide binding do not control mTORC2 activity directly but rather have roles in folding or ternary interactions. These insights provide a firm basis for studying mTORC2 signaling and for developing mTORC2-specific inhibitors.
History
DepositionJul 28, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,157,32718
Polymers1,153,6958
Non-polymers3,63210
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area44160 Å2
ΔGint-206 kcal/mol
Surface area320140 Å2
MethodPISA

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Components

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Protein , 2 types, 4 molecules ABEF

#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 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P42345, non-specific serine/threonine protein kinase
#3: Protein Rapamycin-insensitive companion of mTOR / AVO3 homolog / hAVO3


Mass: 192472.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RICTOR, KIAA1999 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6R327

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Target of rapamycin complex ... , 2 types, 4 molecules CDGH

#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: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BVC4
#4: Protein Target of rapamycin complex 2 subunit MAPKAP1 / TORC2 subunit MAPKAP1 / Mitogen-activated protein kinase 2-associated protein 1 / Stress-activated ...TORC2 subunit MAPKAP1 / Mitogen-activated protein kinase 2-associated protein 1 / Stress-activated map kinase-interacting protein 1 / mSIN1


Mass: 59206.738 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: MAFLDNPTIILAHIRQSHVTSDDTGMCEMVLIDHDVDLEKIHPPSMPGDSGSEIQGSNGE TQGYVYAQSVDITSSWDFGIRRRSNTAQRLERLRKERQNQIKCKNIQWKERNSKQSAQEL KSLFEKKSLKEKPPISGKQSILSVRLEQCPLQLNNPFNEYSKFDGKGHVGTTATKKIDVY ...Details: MAFLDNPTIILAHIRQSHVTSDDTGMCEMVLIDHDVDLEKIHPPSMPGDSGSEIQGSNGE TQGYVYAQSVDITSSWDFGIRRRSNTAQRLERLRKERQNQIKCKNIQWKERNSKQSAQEL KSLFEKKSLKEKPPISGKQSILSVRLEQCPLQLNNPFNEYSKFDGKGHVGTTATKKIDVY LPLHSSQDRLLPMTVVTMASARVQDLIGLICWQYTSEGREPKLNDNVSAYCLHIAEDDGE VDTDFPPLDSNEPIHKFGFSTLALVEKYSSPGLTSKESLFVRINAAHGFSLIQVDNTKVT MKEILLKAVKRRKGSQKVSGPQYRLEKQSEPNVAVDLDSTLESQSAWEFCLVRENSSRAD GVFEEDSQIDIATVQDMLSSHHYKSFKVSMIHRLRFTTDVQLGISGDKVEIDPVTNQKAS TKFWIKQKPISIDSDLLCACDLAEEKSPSHAIFKLTYLSNHDYKHLYFESDAATVNEIVL KVNYILESRASTARADYFAQKQRKLNRRTSFSFQKEKKSGQQ
Source: (gene. exp.) Homo sapiens (human) / Gene: MAPKAP1, MIP1, SIN1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BPZ7

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Non-polymers , 4 types, 10 molecules

#5: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H18O24P6 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-ACE / ACETYL GROUP


Mass: 44.053 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H4O

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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

ComponentName: human mTOR complex 2 / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 1.15 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8.5
SpecimenConc.: 0.37 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 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 / C2 aperture diameter: 100 µm
Image recordingElectron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: EPU / Version: 2 / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 293038 / Symmetry type: POINT

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