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- EMDB-2990: Structure of Target Of Rapapmycin Complex 2 (TORC2) from Saccharo... -

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Basic information

Entry
Database: EMDB / ID: EMD-2990
TitleStructure of Target Of Rapapmycin Complex 2 (TORC2) from Saccharomyces cerevisiae
Map dataReconstruction of yeast TORC2 - filtered at 26 Angstroms resolution (FSC 0.143 criterion after gold standard refinement). Contour level provided by author - The map was generated from negative stain data, and we determined the correct contour level to display the map to be 2.9 in Pymol.
Sample
  • Sample: Yeast Target of Rapamycin Complex 2
  • Protein or peptide: Yeast TORC2CRTC2
KeywordsTOR kinase complex / TORC2 / cell growth / rapamycin
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsGaubitz C / Oliveira TM / Prouteau M / Leitner A / Karuppasamy M / Konstantinidou G / Rispal D / Eltschinger S / Robinson GC / Thore S ...Gaubitz C / Oliveira TM / Prouteau M / Leitner A / Karuppasamy M / Konstantinidou G / Rispal D / Eltschinger S / Robinson GC / Thore S / Aebersold R / Schaffitzel C / Loewith R
CitationJournal: 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 / ...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.
History
DepositionApr 29, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseJun 10, 2015-
UpdateJul 1, 2015-
Current statusJul 1, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.029
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2990.tif

Downloads & links

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Map

FileDownload / File: emd_2990.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of yeast TORC2 - filtered at 26 Angstroms resolution (FSC 0.143 criterion after gold standard refinement). Contour level provided by author - The map was generated from negative stain data, and we determined the correct contour level to display the map to be 2.9 in Pymol.
Voxel sizeX=Y=Z: 4.5 Å
Density
Contour LevelBy AUTHOR: 0.029 / Movie #1: 0.029
Minimum - Maximum-0.0818112 - 0.11751353
Average (Standard dev.)0.00042925 (±0.00938699)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 450.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.54.54.5
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z450.000450.000450.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.0820.1180.000

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Supplemental data

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Sample components

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Entire : Yeast Target of Rapamycin Complex 2

EntireName: Yeast Target of Rapamycin Complex 2
Components
  • Sample: Yeast Target of Rapamycin Complex 2
  • Protein or peptide: Yeast TORC2CRTC2

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Supramolecule #1000: Yeast Target of Rapamycin Complex 2

SupramoleculeName: Yeast Target of Rapamycin Complex 2 / type: sample / ID: 1000 / Details: TORC2 complex, which consists of six subunits / Oligomeric state: dimer / Number unique components: 1
Molecular weightExperimental: 1.4 MDa / Theoretical: 1.4 MDa / Method: Size exclusion

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Macromolecule #1: Yeast TORC2

MacromoleculeName: Yeast TORC2 / type: protein_or_peptide / ID: 1
Details: TORC2 is composed of two copies of: Tor2 kinase, Lst8, Avo1, Avo2, Avo3, Bit61/2
Number of copies: 1 / Oligomeric state: 2 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Location in cell: Plasma membrane MCT (membrane compartment containing TORC2) domain
Molecular weightExperimental: 1.4 MDa / Theoretical: 1.4 MDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5
Details: 50mM HEPES pH 7.5, 5 mM CHAPS, 300 mM KCl, 0,5 mM DTT
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for 1 min.
GridDetails: 300 mesh grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM120T
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 26000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 45
DateApr 10, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 200 / Details: 200 micrographs (100 tilt pairs)
Tilt angle min0

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Image processing

CTF correctionDetails: each micrograph
Final two d classificationNumber classes: 500
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: Xmipp, Relion / Number images used: 24979
DetailsA total of 8,762 RCT pairs were picked manually with tiltpicker (Voss et al, 2009). The additional untilted particles were picked with e2boxer.py (Ludtke, 2010). The 3D reconstruction was calculated with the Xmipp ML tomo and refined with Xmipp MLF 3D (Scheres et al., 2008)

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