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- EMDB-2990: Structure of Target Of Rapapmycin Complex 2 (TORC2) from Saccharo... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2990 | |||||||||
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Title | Structure of Target Of Rapapmycin Complex 2 (TORC2) from Saccharomyces cerevisiae | |||||||||
![]() | Reconstruction 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. | |||||||||
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![]() | TOR kinase complex / TORC2 / cell growth / rapamycin | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 26.0 Å | |||||||||
![]() | Gaubitz 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 | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 3.6 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 10 KB 10 KB | Display Display | ![]() |
Images | ![]() | 739.6 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction 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 size | X=Y=Z: 4.5 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Yeast Target of Rapamycin Complex 2
Entire | Name: Yeast Target of Rapamycin Complex 2 |
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Components |
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-Supramolecule #1000: Yeast Target of Rapamycin Complex 2
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 1.4 MDa / Theoretical: 1.4 MDa / Method: Size exclusion |
-Macromolecule #1: Yeast TORC2
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() Location in cell: Plasma membrane MCT (membrane compartment containing TORC2) domain |
Molecular weight | Experimental: 1.4 MDa / Theoretical: 1.4 MDa |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 7.5 Details: 50mM HEPES pH 7.5, 5 mM CHAPS, 300 mM KCl, 0,5 mM DTT |
Staining | Type: NEGATIVE Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for 1 min. |
Grid | Details: 300 mesh grid |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
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Electron microscopy
Microscope | FEI/PHILIPS CM120T |
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Date | Apr 10, 2012 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 200 / Details: 200 micrographs (100 tilt pairs) |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 26000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 45 |
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Image processing
Details | A 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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CTF correction | Details: each micrograph |
Final reconstruction | Applied 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 |
Final two d classification | Number classes: 500 |