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- EMDB-3896: Cryo-EM Structure of Saccharomyces cerevisiae Target of Rapamycin... -

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Entry
Database: EMDB / ID: 3896
TitleCryo-EM Structure of Saccharomyces cerevisiae Target of Rapamycin Complex 2
Map datarefined and postprocessed map
SampleTarget of rapamycin protein complex 2MTOR
  • Serine/threonine-protein kinase TOR2
  • Target of rapamycin complex subunit LST8MTOR
  • (Target of rapamycin complex 2 subunit ...) x 3
Function / homologyFATC domain / WD40-repeat-containing domain / WD40-repeat-containing domain superfamily / Sin1, middle CRIM domain / SAPK-interacting protein 1, Pleckstrin-homology domain / Serine/threonine-protein kinase TOR / Domain of unknown function DUF3385, target of rapamycin protein / Ankyrin repeat-containing domain / G-protein beta WD-40 repeat / WD40 repeat, conserved site ...FATC domain / WD40-repeat-containing domain / WD40-repeat-containing domain superfamily / Sin1, middle CRIM domain / SAPK-interacting protein 1, Pleckstrin-homology domain / Serine/threonine-protein kinase TOR / Domain of unknown function DUF3385, target of rapamycin protein / Ankyrin repeat-containing domain / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Phosphatidylinositol 3/4-kinase, conserved site / Armadillo-type fold / Ankyrin repeat-containing domain superfamily / Phosphatidylinositol 3-/4-kinase, catalytic domain / WD40 repeat / Ankyrin repeat / PIK-related kinase, FAT / WD40/YVTN repeat-like-containing domain superfamily / FKBP12-rapamycin binding domain / Protein kinase-like domain superfamily / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / PIK-related kinase / FKBP12-rapamycin binding domain superfamily / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Target of rapamycin complex subunit LST8 / HSF1-dependent transactivation / FATC domain profile. / FAT domain profile. / Ankyrin repeat region circular profile. / Trp-Asp (WD) repeats circular profile. / Phosphatidylinositol 3- and 4-kinases family profile. / Ankyrin repeat profile. / Trp-Asp (WD) repeats profile. / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3- and 4-kinases signature 1. / Trp-Asp (WD) repeats signature. / SAPK-interacting protein 1 (Sin1), Pleckstrin-homology / SAPK-interacting protein 1 (Sin1), middle CRIM domain / Domain of unknown function (DUF3385) / FKBP12-rapamycin binding domain / FATC domain / FAT domain / Phosphatidylinositol 3- and 4-kinase / WD domain, G-beta repeat / Ankyrin repeat / PH-like domain superfamily / actin filament reorganization involved in cell cycle / regulation of snRNA pseudouridine synthesis / mitochondria-nucleus signaling pathway / TORC2 signaling / positive regulation of Rho guanyl-nucleotide exchange factor activity / 1-phosphatidylinositol 4-kinase / 1-phosphatidylinositol 4-kinase activity / establishment or maintenance of actin cytoskeleton polarity / Ras GTPase binding / TORC1 complex / TORC2 complex / fungal-type cell wall organization / fungal-type vacuole membrane / positive regulation of TOR signaling / vacuolar membrane / protein kinase activator activity / negative regulation of Ras protein signal transduction / positive regulation of Rho protein signal transduction / positive regulation of endocytosis / TOR signaling / cytoskeleton organization / stress-activated protein kinase signaling cascade / nuclear periphery / regulation of actin cytoskeleton organization / phosphatidylinositol-4,5-bisphosphate binding / negative regulation of autophagy / regulation of cell growth / regulation of cell cycle / ribosome biogenesis / extrinsic component of cytoplasmic side of plasma membrane / endosome membrane / protein-containing complex binding / non-specific serine/threonine protein kinase / Golgi membrane / DNA repair / protein phosphorylation / protein serine/threonine kinase activity / signal transduction / ATP binding / plasma membrane / nucleus / cytoplasm / Serine/threonine-protein kinase TOR2 / Target of rapamycin complex subunit LST8 / Target of rapamycin complex 2 subunit AVO2 / Target of rapamycin complex 2 subunit AVO1
Function and homology information
SourceSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / 7.9 Å resolution
AuthorsKaruppasamy M / Kusmider B / Oliveira TM / Gaubitz C / Prouteau M / Loewith R / Schaffitzel C
Citation
Journal: Nat Commun / Year: 2017
Title: Cryo-EM structure of Saccharomyces cerevisiae target of rapamycin complex 2.
Authors: Manikandan Karuppasamy / Beata Kusmider / Taiana M Oliveira / Christl Gaubitz / Manoel Prouteau / Robbie Loewith / Christiane Schaffitzel
#1: Journal: Mol. Cell / Year: 2015
Title: Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2.
Authors: Christl Gaubitz / Taiana M Oliveira / Manoel Prouteau / Alexander Leitner / Manikandan Karuppasamy / Georgia Konstantinidou / Delphine Rispal / Sandra Eltschinger / Graham C Robinson / Stéphane Thore / Ruedi Aebersold / Christiane Schaffitzel / Robbie Loewith
Abstract: Target of Rapamycin (TOR) plays central roles in the regulation of eukaryote growth as the hub of two essential multiprotein complexes: TORC1, which is rapamycin-sensitive, and the lesser ...Target of Rapamycin (TOR) plays central roles in the regulation of eukaryote growth as the hub of two essential multiprotein complexes: TORC1, which is rapamycin-sensitive, and the lesser characterized TORC2, which is not. TORC2 is a key regulator of lipid biosynthesis and Akt-mediated survival signaling. In spite of its importance, its structure and the molecular basis of its rapamycin insensitivity are unknown. Using crosslinking-mass spectrometry and electron microscopy, we determined the architecture of TORC2. TORC2 displays a rhomboid shape with pseudo-2-fold symmetry and a prominent central cavity. Our data indicate that the C-terminal part of Avo3, a subunit unique to TORC2, is close to the FKBP12-rapamycin-binding domain of Tor2. Removal of this sequence generated a FKBP12-rapamycin-sensitive TORC2 variant, which provides a powerful tool for deciphering TORC2 function in vivo. Using this variant, we demonstrate a role for TORC2 in G2/M cell-cycle progression.
Validation ReportPDB-ID: 6emk

SummaryFull reportAbout validation report
DateDeposition: Oct 2, 2017 / Header (metadata) release: Oct 18, 2017 / Map release: Dec 6, 2017 / Last update: Oct 17, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0147
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0147
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6emk
  • Surface level: 0.0147
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6emk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3896.map.gz (map file in CCP4 format, 131073 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1.38 Å/pix.
= 441.6 Å
320 pix
1.38 Å/pix.
= 441.6 Å
320 pix
1.38 Å/pix.
= 441.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.38 Å
Density
Contour Level:0.0147 (by author), 0.0147 (movie #1):
Minimum - Maximum-0.043736793 - 0.10092282
Average (Standard dev.)0.0011989143 (0.0065768785)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin0.00.00.0
Limit319.0319.0319.0
Spacing320320320
CellA=B=C: 441.6 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.381.381.38
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z441.600441.600441.600
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0440.1010.001

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Supplemental data

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Sample components

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Entire Target of rapamycin protein complex 2

EntireName: Target of rapamycin protein complex 2 / Number of components: 6

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Component #1: protein, Target of rapamycin protein complex 2

ProteinName: Target of rapamycin protein complex 2MTOR / Recombinant expression: No
MassTheoretical: 1.4 MDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, Serine/threonine-protein kinase TOR2

ProteinName: Serine/threonine-protein kinase TOR2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 281.915438 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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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: 34.077879 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #4: protein, Target of rapamycin complex 2 subunit TSC11

ProteinName: Target of rapamycin complex 2 subunit TSC11 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 25.804662 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #5: protein, Target of rapamycin complex 2 subunit AVO2

ProteinName: Target of rapamycin complex 2 subunit AVO2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 47.206457 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #6: protein, Target of rapamycin complex 2 subunit AVO1

ProteinName: Target of rapamycin complex 2 subunit AVO1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 131.565453 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 4 K / Humidity: 100 % / Details: 2 - 3 sec blotting.

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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: 47 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 105000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 3500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 70.0 - 70.0 K)
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image acquisition

Image acquisition #1Number of digital images: 4189
Image acquisition #2Number of digital images: 2847

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 16190
3D reconstruction #1Software: RELION / Resolution: 7.9 Å / Resolution method: FSC 0.143 CUT-OFF
3D reconstruction #2Software: RELION / Resolution: 8 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 5FVM
Output model

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