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- EMDB-11488: cryo-EM structure of human mTOR complex 2, overall refinement -

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Basic information

Entry
Database: EMDB / ID: EMD-11488
Titlecryo-EM structure of human mTOR complex 2, overall refinement
Map data
Samplehuman mTOR complex 2MTORC2
  • Serine/threonine-protein kinase mTOR
  • (Target of rapamycin complex ...MTOR) x 2
  • Rapamycin-insensitive companion of mTOR
  • (ligand) x 4
Function / homology
Function and homology information


TORC2 signaling / regulation of peptidyl-serine phosphorylation / establishment or maintenance of actin cytoskeleton polarity / positive regulation of wound healing, spreading of epidermal cells / positive regulation of skeletal muscle hypertrophy / negative regulation of iodide transmembrane transport / positive regulation of cytoplasmic translational initiation / positive regulation of granulosa cell proliferation / negative regulation of muscle atrophy / negative regulation of cholangiocyte apoptotic process ...TORC2 signaling / regulation of peptidyl-serine phosphorylation / establishment or maintenance of actin cytoskeleton polarity / positive regulation of wound healing, spreading of epidermal cells / positive regulation of skeletal muscle hypertrophy / negative regulation of iodide transmembrane transport / positive regulation of cytoplasmic translational initiation / positive regulation of granulosa cell proliferation / negative regulation of muscle atrophy / negative regulation of cholangiocyte apoptotic process / regulation of cellular response to oxidative stress / regulation of fatty acid beta-oxidation / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cholangiocyte proliferation / RNA polymerase III type 2 promoter sequence-specific DNA binding / positive regulation of eating behavior / regulation of locomotor rhythm / regulation of carbohydrate utilization / cardiac muscle cell development / regulation of response to food / cellular response to leucine starvation / nucleus localization / cellular response to leucine / TFIIIC-class transcription factor complex binding / positive regulation of sensory perception of pain / heart valve morphogenesis / small GTPase binding => GO:0031267 / phosphatidic acid binding / RNA polymerase III type 3 promoter sequence-specific DNA binding / regulation of membrane permeability / regulation of brown fat cell differentiation / TORC1 complex / TORC1 signaling / TORC2 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / voluntary musculoskeletal movement / regulation of protein kinase B signaling / regulation of osteoclast differentiation / regulation of glycogen biosynthetic process / energy reserve metabolic process / positive regulation of keratinocyte migration / positive regulation of neuron maturation / regulation of phosphorylation / positive regulation of transcription by RNA polymerase III / negative regulation of cell size / ruffle organization / embryo development ending in birth or egg hatching / lysosome organization / negative regulation of Ras protein signal transduction / anoikis / positive regulation of TOR signaling / cellular response to nutrient levels / phosphatidylinositol-3,5-bisphosphate binding / mRNA stabilization / positive regulation of glial cell proliferation / phosphatidylinositol-3,4-bisphosphate binding / positive regulation of cell growth involved in cardiac muscle cell development / regulation of establishment of cell polarity / regulation of myelination / positive regulation of dendritic spine development / positive regulation of lamellipodium assembly / negative regulation of calcineurin-NFAT signaling cascade / negative regulation of macroautophagy / postsynaptic cytosol / spinal cord development / regulation of cell size / germ cell development / maternal process involved in female pregnancy / enzyme activator activity / TOR signaling / positive regulation of myotube differentiation / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of actin filament polymerization / positive regulation of epithelial to mesenchymal transition / social behavior / positive regulation of lipid biosynthetic process / cardiac muscle contraction / protein catabolic process / positive regulation of oligodendrocyte differentiation / long-term memory / response to morphine / positive regulation of phosphoprotein phosphatase activity / heart morphogenesis / regulation of macroautophagy / endomembrane system / cellular response to amino acid starvation / regulation of GTPase activity / regulation of actin cytoskeleton organization / positive regulation of stress fiber assembly / substantia nigra development / response to insulin / response to cocaine / 'de novo' pyrimidine nucleobase biosynthetic process / cell aging / response to amino acid / actin cytoskeleton reorganization / phagocytic vesicle / negative regulation of protein ubiquitination / phosphorylation / establishment of cell polarity
Protein kinase-like domain superfamily / PIK-related kinase / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Pianissimo family / Rapamycin-insensitive companion of mTOR, N-terminal domain / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / PH-like domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Rapamycin-insensitive companion of mTOR, domain 4 ...Protein kinase-like domain superfamily / PIK-related kinase / Rapamycin-insensitive companion of mTOR, phosphorylation-site / Pianissimo family / Rapamycin-insensitive companion of mTOR, N-terminal domain / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / PH-like domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Rapamycin-insensitive companion of mTOR, domain 4 / Quinoprotein alcohol dehydrogenase-like superfamily / Armadillo-type fold / WD40-repeat-containing domain / Phosphatidylinositol 3/4-kinase, conserved site / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Serine/threonine-protein kinase TOR / Rapamycin-insensitive companion of mTOR, domain 5 / Rapamycin-insensitive companion of mTOR, middle domain / SAPK-interacting protein 1, Pleckstrin-homology domain / Sin1, N-terminal / FKBP12-rapamycin binding domain / FATC domain / PIK-related kinase, FAT / WD40 repeat / Target of rapamycin complex subunit LST8 / Phosphatidylinositol 3-/4-kinase, catalytic domain / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / FKBP12-rapamycin binding domain superfamily / Domain of unknown function DUF3385, target of rapamycin protein / Sin1, middle CRIM domain
Serine/threonine-protein kinase mTOR / Rapamycin-insensitive companion of mTOR / Target of rapamycin complex 2 subunit MAPKAP1 / Target of rapamycin complex subunit LST8
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsScaiola A / Mangia F / Imseng S / Boehringer D / Ban N / Maier T
Funding support Switzerland, 3 items
OrganizationGrant numberCountry
Swiss National Science Foundation179323 Switzerland
Swiss National Science Foundation138262
Swiss National Science Foundation177084 Switzerland
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.
Validation ReportPDB-ID: 6zwm

SummaryFull reportAbout validation report
History
DepositionJul 28, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateNov 18, 2020-
Current statusNov 18, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.375
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.375
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zwm
  • Surface level: 0.375
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11488.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 320 pix.
= 430.08 Å
1.34 Å/pix.
x 320 pix.
= 430.08 Å
1.34 Å/pix.
x 320 pix.
= 430.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.344 Å
Density
Contour LevelBy AUTHOR: 0.375 / Movie #1: 0.375
Minimum - Maximum-1.7557929 - 3.3231337
Average (Standard dev.)-0.0021909792 (±0.07714291)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 430.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3441.3441.344
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z430.080430.080430.080
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-1.7563.323-0.002

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Supplemental data

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Segmentation: #1

Fileemd_11488_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Segmentation: #2

Fileemd_11488_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_11488_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_11488_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire human mTOR complex 2

EntireName: human mTOR complex 2MTORC2 / Number of components: 9

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Component #1: protein, human mTOR complex 2

ProteinName: human mTOR complex 2MTORC2 / Recombinant expression: No
MassTheoretical: 1.15 MDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #2: protein, Serine/threonine-protein kinase mTOR

ProteinName: Serine/threonine-protein kinase mTOR / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 289.257969 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, Target of rapamycin complex subunit LST8

ProteinName: Target of rapamycin complex subunit LST8MTOR / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 35.91009 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #4: protein, Rapamycin-insensitive companion of mTOR

ProteinName: Rapamycin-insensitive companion of mTOR / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 192.472922 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #5: protein, Target of rapamycin complex 2 subunit MAPKAP1

ProteinName: Target of rapamycin complex 2 subunit MAPKAP1
Details: MAFLDNPTIILAHIRQSHVTSDDTGMCEMVLIDHDVDLEKIHPPSMPGDSGSEIQGSNGE TQGYVYAQSVDITSSWDFGIRRRSNTAQRLERLRKERQNQIKCKNIQWKERNSKQSAQEL KSLFEKKSLKEKPPISGKQSILSVRLEQCPLQLNNPFNEYSKFDGKGHVGTTATKKIDVY ...Details: MAFLDNPTIILAHIRQSHVTSDDTGMCEMVLIDHDVDLEKIHPPSMPGDSGSEIQGSNGE TQGYVYAQSVDITSSWDFGIRRRSNTAQRLERLRKERQNQIKCKNIQWKERNSKQSAQEL KSLFEKKSLKEKPPISGKQSILSVRLEQCPLQLNNPFNEYSKFDGKGHVGTTATKKIDVY LPLHSSQDRLLPMTVVTMASARVQDLIGLICWQYTSEGREPKLNDNVSAYCLHIAEDDGE VDTDFPPLDSNEPIHKFGFSTLALVEKYSSPGLTSKESLFVRINAAHGFSLIQVDNTKVT MKEILLKAVKRRKGSQKVSGPQYRLEKQSEPNVAVDLDSTLESQSAWEFCLVRENSSRAD GVFEEDSQIDIATVQDMLSSHHYKSFKVSMIHRLRFTTDVQLGISGDKVEIDPVTNQKAS TKFWIKQKPISIDSDLLCACDLAEEKSPSHAIFKLTYLSNHDYKHLYFESDAATVNEIVL KVNYILESRASTARADYFAQKQRKLNRRTSFSFQKEKKSGQQ
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 59.206738 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #6: ligand, PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

LigandName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.523247 kDa

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Component #7: ligand, INOSITOL HEXAKISPHOSPHATE

LigandName: INOSITOL HEXAKISPHOSPHATEPhytic acid / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.660035 kDa

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Component #8: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #9: ligand, ACETYL GROUP

LigandName: ACETYL GROUP / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 4.405305 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.37 mg/mL / pH: 8.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: OTHER / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 70 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 293038
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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