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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 / homology
Function and homology information


PIP3 activates AKT signaling / CD28 dependent PI3K/Akt signaling / regulation of snRNA pseudouridine synthesis / mitochondria-nucleus signaling pathway / Regulation of TP53 Degradation / positive regulation of Rho guanyl-nucleotide exchange factor activity / TORC2 signaling / 1-phosphatidylinositol 4-kinase / 1-phosphatidylinositol 4-kinase activity / establishment or maintenance of actin cytoskeleton polarity ...PIP3 activates AKT signaling / CD28 dependent PI3K/Akt signaling / regulation of snRNA pseudouridine synthesis / mitochondria-nucleus signaling pathway / Regulation of TP53 Degradation / positive regulation of Rho guanyl-nucleotide exchange factor activity / TORC2 signaling / 1-phosphatidylinositol 4-kinase / 1-phosphatidylinositol 4-kinase activity / establishment or maintenance of actin cytoskeleton polarity / VEGFR2 mediated vascular permeability / HSF1-dependent transactivation / fungal-type cell wall organization / TORC2 complex / Amino acids regulate mTORC1 / TORC1 complex / fungal-type vacuole membrane / vacuolar membrane / positive regulation of Rho protein signal transduction / negative regulation of macroautophagy / positive regulation of endocytosis / TOR signaling / cytoskeleton organization / phosphatidylinositol-4,5-bisphosphate binding / nuclear periphery / negative regulation of autophagy / response to nutrient / regulation of cell growth / regulation of actin cytoskeleton organization / ribosome biogenesis / endosome membrane / regulation of cell cycle / non-specific serine/threonine protein kinase / cell cycle / Golgi membrane / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / protein-containing complex binding / signal transduction / mitochondrion / ATP binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
TORC2 component Sin1/Avo1 / SAPK-interacting protein 1, Pleckstrin-homology domain / Sin1, middle CRIM domain / SAPK-interacting protein 1 (Sin1), middle CRIM domain / SAPK-interacting protein 1 (Sin1), Pleckstrin-homology / Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3447 / Domain of unknown function DUF3385, target of rapamycin protein / Domain of unknown function (DUF3385) / Domain of unknown function ...TORC2 component Sin1/Avo1 / SAPK-interacting protein 1, Pleckstrin-homology domain / Sin1, middle CRIM domain / SAPK-interacting protein 1 (Sin1), middle CRIM domain / SAPK-interacting protein 1 (Sin1), Pleckstrin-homology / Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3447 / Domain of unknown function DUF3385, target of rapamycin protein / Domain of unknown function (DUF3385) / Domain of unknown function / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR / FKBP12-rapamycin binding domain superfamily / FKBP12-rapamycin binding domain / PIK-related kinase, FAT / FAT domain / FATC domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Ankyrin repeat / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / PH-like domain superfamily / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
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
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.9 Å
AuthorsKaruppasamy, M. / Kusmider, B. / Oliveira, T.M. / Gaubitz, C. / Prouteau, M. / Loewith, R. / Schaffitzel, C.
Funding support4items
OrganizationGrant numberCountry
Swiss National Science FoundationFNS 31003A_160023
Sinergia grantCRSII3_136254
European Research CouncilTORCH 614552
European Research CouncilStarting Grant, No 281331
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 /
Abstract: The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not ...The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not inhibited by the macrolide rapamycin. Here, we present the structure of Saccharomyces cerevisiae TORC2 determined by electron cryo-microscopy. TORC2 contains six subunits assembling into a 1.4 MDa rhombohedron. Tor2 and Lst8 form the common core of both TOR complexes. Avo3/Rictor is unique to TORC2, but interacts with the same HEAT repeats of Tor2 that are engaged by Kog1/Raptor in mammalian TORC1, explaining the mutual exclusivity of these two proteins. Density, which we conclude is Avo3, occludes the FKBP12-rapamycin-binding site of Tor2's FRB domain rendering TORC2 rapamycin insensitive and recessing the kinase active site. Although mobile, Avo1/hSin1 further restricts access to the active site as its conserved-region-in-the-middle (CRIM) domain is positioned along an edge of the TORC2 active-site-cleft, consistent with a role for CRIM in substrate recruitment.
#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 / ...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
DepositionOct 2, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 6, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 17, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.2Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • Deposited structure unit
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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
Polymers1,041,14010
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, final cryoEM map
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16320 Å2
ΔGint-69 kcal/mol
Surface area292380 Å2
MethodPISA

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Components

#1: Protein Serine/threonine-protein kinase TOR2 / Dominant rapamycin resistance protein 2 / Phosphatidylinositol 4-kinase TOR2 / PtdIns-4-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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: TOR2, DRR2, TSC14, YKL203C / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P32600, 1-phosphatidylinositol 4-kinase, non-specific serine/threonine protein kinase
#2: Protein Target of rapamycin complex subunit LST8 / MTOR / TORC subunit LST8 / Lethal with SEC13 protein 8


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


Mass: 25804.662 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast)
#4: Protein 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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: AVO2, YMR068W, YM9916.07 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04749
#5: Protein 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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: AVO1, YOL078W, O1110 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08236

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Target of rapamycin protein complex 2MTOR / Type: COMPLEX / Entity ID: all / 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 (brewer'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 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K / Details: 2 - 3 sec blotting

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recording

Imaging-ID: 1

IDAverage exposure time (sec.)Electron dose (e/Å2)Detector modeFilm or detector modelNum. of grids imagedNum. 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-IDNum. of particles selected
11350000
22200000
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
116EMKC2 (2 fold cyclic)
226EMKC2 (2 fold cyclic)
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
17.9FSC 0.143 CUT-OFF1619016EMKPOINT
28FSC 0.143 CUT-OFF1066326EMKPOINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5FVM

5fvm


Pdb chain residue range: 81-2474
RefinementResolution: 8→220.8 Å / Cor.coef. Fo:Fc: 0.987 / SU B: 111.293 / SU ML: 0.794
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.12139 --
obs0.12139 30863 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 295.421 Å2
Baniso -1Baniso -2Baniso -3
1--0.8 Å22.16 Å2-1.12 Å2
2---1.83 Å20.11 Å2
3---2.63 Å2
Refinement stepCycle: 1 / Total: 48016
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0130.0247333
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg2.0241.9661663
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg10.87654749
ELECTRON MICROSCOPYr_dihedral_angle_2_deg40.82824.4012036
ELECTRON MICROSCOPYr_dihedral_angle_3_deg23.376158088
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.61915268
ELECTRON MICROSCOPYr_chiral_restr0.1530.27744
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_it19.39445.9926911
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_refined47.6478872
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 8→8.207 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.377 2391 -
Rfree-0 -
obs--100 %

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