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- PDB-6emk: Cryo-EM Structure of Saccharomyces cerevisiae Target of Rapamycin... -

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Basic information

Entry
Database: PDB / ID: 6emk
TitleCryo-EM Structure of Saccharomyces cerevisiae Target of Rapamycin Complex 2
Components
  • Serine/threonine-protein kinase TOR2
  • Target of rapamycin complex 2 subunit AVO1
  • Target of rapamycin complex 2 subunit AVO2
  • Target of rapamycin complex 2 subunit TSC11
  • Target of rapamycin complex subunit LST8MTOR
KeywordsSIGNALING PROTEIN / target of rapamycin / torc2 / FRB domain / Tor2-Lst8 / kinases
Function / homologyAnkyrin repeats (many copies) / WD40 repeat, conserved site / FKBP12-rapamycin binding domain superfamily / 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 ...Ankyrin repeats (many copies) / WD40 repeat, conserved site / FKBP12-rapamycin binding domain superfamily / 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 / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / WD40-repeat-containing domain / Armadillo-type fold / WD40/YVTN repeat-like-containing domain superfamily / PIK-related kinase / PH-like domain superfamily / Tetratricopeptide-like helical domain superfamily / Armadillo-like helical / Protein kinase-like domain superfamily / Ankyrin repeat-containing domain superfamily / Target of rapamycin complex subunit LST8 / PIK-related kinase, FAT / Phosphatidylinositol 3- and 4-kinases signature 2. / 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 1. / Ankyrin repeat / 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 / FATC domain / FKBP12-rapamycin binding domain / Ankyrin repeat / Phosphatidylinositol 3-/4-kinase, catalytic domain / WD40 repeat / 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 activity / 1-phosphatidylinositol 4-kinase / 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
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 7.9 Å resolution
AuthorsKaruppasamy, M. / Kusmider, B. / Oliveira, T.M. / Gaubitz, C. / Prouteau, M. / Loewith, R. / Schaffitzel, C.
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 / 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 Report
SummaryFull reportAbout validation report
DateDeposition: Oct 2, 2017 / Release: Dec 6, 2017
RevisionDateData content typeGroupCategoryProviderType
1.0Dec 6, 2017Structure modelrepositoryInitial release
1.1Oct 17, 2018Structure modelData collection / Refinement descriptionrefine

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Serine/threonine-protein kinase TOR2
B: Target of rapamycin complex subunit LST8
C: Serine/threonine-protein kinase TOR2
D: Target of rapamycin complex subunit LST8
E: Target of rapamycin complex 2 subunit TSC11
F: Target of rapamycin complex 2 subunit TSC11
G: Target of rapamycin complex 2 subunit AVO2
H: Target of rapamycin complex 2 subunit AVO2
I: Target of rapamycin complex 2 subunit AVO1
J: Target of rapamycin complex 2 subunit AVO1


Theoretical massNumber of molelcules
Total (without water)1,041,14010
Polyers1,041,14010
Non-polymers00
Water0
1


  • idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, final cryoEM map
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)16320
ΔGint (kcal/M)-69
Surface area (Å2)292380
MethodPISA

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Components

#1: Protein/peptide Serine/threonine-protein kinase TOR2 / Dominant rapamycin resistance protein 2 / Phosphatidylinositol 4-kinase TOR2 / PtdIns-4-kinase TOR2 / Target of rapamycin kinase 2 / Temperature-sensitive CSG2 suppressor protein 14


Mass: 281915.438 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: TOR2, DRR2, TSC14, YKL203C / Production host: Saccharomyces cerevisiae (baker's yeast)
References: UniProt: P32600, 1-phosphatidylinositol 4-kinase, non-specific serine/threonine protein kinase
#2: Protein/peptide Target of rapamycin complex subunit LST8 / MTOR / TORC subunit LST8 / Lethal with SEC13 protein 8


Mass: 34077.879 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: LST8, YNL006W, N2005 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P41318
#3: Protein/peptide Target of rapamycin complex 2 subunit TSC11


Mass: 25804.662 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Production host: Saccharomyces cerevisiae (baker's yeast)
#4: Protein/peptide Target of rapamycin complex 2 subunit AVO2 / TORC2 subunit AVO2 / Adheres voraciously to TOR2 protein 2


Mass: 47206.457 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: AVO2, YMR068W, YM9916.07 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q04749
#5: Protein/peptide Target of rapamycin complex 2 subunit AVO1 / TORC2 subunit AVO1 / Adheres voraciously to TOR2 protein 1


Mass: 131565.453 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: AVO1, YOL078W, O1110 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q08236

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Target of rapamycin protein complex 2MTOR / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5 / Source: RECOMBINANT
Molecular weightValue: 1.4 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 kelvins / Details: 2 - 3 sec blotting

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 kelvins / Temperature (min): 70 kelvins
Image recording

Imaging ID: 1

IDAverage exposure timeElectron doseDetector modeFilm or detector modelNumber of grids imagedNumber of real images
12.350INTEGRATINGFEI FALCON II (4k x 4k)44189
22047SUPER-RESOLUTIONGATAN K2 QUANTUM (4k x 4k)12847
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameVersionCategoryImage processing ID
2SerialEM3.6image acquisition
4CTFFIND4CTF correction1
7Coot0.8.8model fitting
9RELION1.4initial Euler assignment1
10RELION1.4final Euler assignment1
12RELION1.43D reconstruction1
13EMAN2particle selection2
15CTFFIND4CTF correction2
16RELION1.4initial Euler assignment2
17RELION1.4final Euler assignment2
19RELION1.43D reconstruction2
20REFMAC5.8.0158model refinement
Image processing
IDImage recording ID
11
22
CTF correction
IDEM image processing IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
IDImage processing IDNumber of particles selected
11350000
22200000
Symmetry
IDImage processing IDEntry IDPoint symmetry
116EMKC2
226EMKC2
3D reconstruction

Entry ID: 6EMK / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDResolutionNumber of particlesImage processing ID
17.9161901
28.0106632
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 5FVM
Pdb chain residue range: 81-2474
RefineCorrelation coeff Fo to Fc: 0.987 / Overall SU B: 111.293 / Overall SU ML: 0.794
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Solvent computationSolvent model details: PARAMETERS FOR MASK CACLULATION
Displacement parametersB iso mean: 295.421 Å2 / Aniso B11: -0.8 Å2 / Aniso B12: 2.16 Å2 / Aniso B13: -1.12 Å2 / Aniso B22: -1.83 Å2 / Aniso B23: 0.11 Å2 / Aniso B33: 2.63 Å2
Least-squares processR factor R work: 0.12139 / R factor obs: 0.12139 / Highest resolution: 8 Å / Lowest resolution: 220.8 Å / Number reflection obs: 30863 / Percent reflection obs: 1
Number of atoms included #1Total: 48016
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0130.02047333
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg2.0241.96061663
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg10.8765.0004749
ELECTRON MICROSCOPYr_dihedral_angle_2_deg40.82824.4012036
ELECTRON MICROSCOPYr_dihedral_angle_3_deg23.37615.0008088
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.61915.000268
ELECTRON MICROSCOPYr_chiral_restr0.1530.2007744
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.02130090
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it10.66830.43623745
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it19.39445.99026911
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it11.15330.10823588
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined47.64078872
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS shellHighest resolution: 8 Å / R factor R work: 0.377 / Lowest resolution: 8.207 Å / Number reflection R free: 0 / Number reflection R work: 2391 / Total number of bins used: 20 / Percent reflection obs: 1

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