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- PDB-7pqh: Cryo-EM structure of Saccharomyces cerevisiae TOROID (TORC1 Organ... -

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Basic information

Entry
Database: PDB / ID: 7pqh
TitleCryo-EM structure of Saccharomyces cerevisiae TOROID (TORC1 Organized in Inhibited Domains).
Components
  • Serine/threonine-protein kinase TOR2
  • Target of rapamycin complex 1 subunit KOG1,Target of rapamycin complex 1 subunit Kog1
  • Target of rapamycin complex subunit LST8MTOR
KeywordsSIGNALING PROTEIN / TARGET OF RAPAMYCIN / TORC1 / KOG1 / LST8 / TOR2 / FRB DOMAIN
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 / 1-phosphatidylinositol 4-kinase / 1-phosphatidylinositol 4-kinase activity / establishment or maintenance of actin cytoskeleton polarity / VEGFR2 mediated vascular permeability ...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 / 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 / cellular response to starvation / nuclear periphery / response to nutrient / negative regulation of autophagy / ubiquitin binding / regulation of autophagy / regulation of cell growth / regulation of actin cytoskeleton organization / cellular response to amino acid stimulus / positive regulation of protein serine/threonine kinase activity / cytoplasmic stress granule / protein-macromolecule adaptor activity / ribosome biogenesis / positive regulation of cell growth / 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
Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / Target of rapamycin complex subunit LST8 / 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 ...Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / Target of rapamycin complex subunit LST8 / 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 / HEAT repeat / HEAT repeat / 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 / Armadillo-like helical / Tetratricopeptide-like helical 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 1 subunit KOG1 / Target of rapamycin complex subunit LST8
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsFelix, J. / Prouteau, M. / Bourgoint, C. / Bonadei, L. / Desfosses, A. / Gabus, C. / Sadian, Y. / Savvides, S.N. / Gutsche, I. / Loewith, R.
Funding supportEuropean Union, 4items
OrganizationGrant numberCountry
European Research Council (ERC)834394European Union
Swiss National Science Foundation179517European Union
H2020 Marie Curie Actions of the European Commission789385European Union
European Research Council (ERC)64778European Union
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: EGOC inhibits TOROID polymerization by structurally activating TORC1.
Authors: Manoël Prouteau / Clélia Bourgoint / Jan Felix / Lenny Bonadei / Yashar Sadian / Caroline Gabus / Savvas N Savvides / Irina Gutsche / Ambroise Desfosses / Robbie Loewith /
Abstract: Target of rapamycin complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. In Saccharomyces cerevisiae, glucose depletion triggers a ...Target of rapamycin complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. In Saccharomyces cerevisiae, glucose depletion triggers a redistribution of TORC1 from a dispersed localization over the vacuole surface into a large, inactive condensate called TOROID (TORC1 organized in inhibited domains). However, the mechanisms governing this transition have been unclear. Here, we show that acute depletion and repletion of EGO complex (EGOC) activity is sufficient to control TOROID distribution, independently of other nutrient-signaling pathways. The 3.9-Å-resolution structure of TORC1 from TOROID cryo-EM data together with interrogation of key interactions in vivo provide structural insights into TORC1-TORC1' and TORC1-EGOC interaction interfaces. These data support a model in which glucose-dependent activation of EGOC triggers binding to TORC1 at an interface required for TOROID assembly, preventing TORC1 polymerization and promoting release of active TORC1.
History
DepositionSep 17, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 29, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Target of rapamycin complex 1 subunit KOG1,Target of rapamycin complex 1 subunit Kog1
B: Target of rapamycin complex 1 subunit KOG1,Target of rapamycin complex 1 subunit Kog1
C: Target of rapamycin complex subunit LST8
D: Target of rapamycin complex subunit LST8
E: Serine/threonine-protein kinase TOR2
F: Serine/threonine-protein kinase TOR2
G: Target of rapamycin complex 1 subunit KOG1,Target of rapamycin complex 1 subunit Kog1
H: Serine/threonine-protein kinase TOR2
I: Target of rapamycin complex subunit LST8
J: Target of rapamycin complex 1 subunit KOG1,Target of rapamycin complex 1 subunit Kog1
K: Serine/threonine-protein kinase TOR2
L: Target of rapamycin complex subunit LST8


Theoretical massNumber of molelcules
Total (without water)1,998,35212
Polymers1,998,35212
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Target of rapamycin complex 1 subunit KOG1,Target of rapamycin complex 1 subunit Kog1 / TORC1 subunit KOG1 / Kontroller of growth protein 1 / Local anesthetic-sensitive protein 24


Mass: 183594.562 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: S. cerevisiae TOROIDS (TORC1 Organized in Inhibited Domains) in the sample are composed of Kog1 (P38873) Lst8, P41318) and Tor2 (P32600),S. cerevisiae TOROIDS (TORC1 Organized in Inhibited ...Details: S. cerevisiae TOROIDS (TORC1 Organized in Inhibited Domains) in the sample are composed of Kog1 (P38873) Lst8, P41318) and Tor2 (P32600),S. cerevisiae TOROIDS (TORC1 Organized in Inhibited Domains) in the sample are composed of Kog1 (P38873) Lst8, P41318) and Tor2 (P32600)
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: KOG1, LAS24, YHR186C, H9998.14 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38873
#2: Protein
Target of rapamycin complex subunit LST8 / MTOR / TORC subunit LST8 / Lethal with SEC13 protein 8


Mass: 34077.879 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: S. cerevisiae TOROIDs (TORC1 Organized in Inhibited Domains) in the sample are composed of Kog1 (P38873), Lst8 (P41318) and Tor2 (P32600)
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: LST8, YNL006W, N2005 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P41318
#3: Protein
Serine/threonine-protein kinase TOR2 / Dominant rapamycin resistance protein 2 / Phosphatidylinositol 4-kinase TOR2 / PI4-kinase TOR2 / ...Dominant rapamycin resistance protein 2 / Phosphatidylinositol 4-kinase TOR2 / PI4-kinase TOR2 / PI4K TOR2 / PtdIns-4-kinase TOR2 / Target of rapamycin kinase 2 / Temperature-sensitive CSG2 suppressor protein 14


Mass: 281915.438 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: S. cerevisiae TOROIDs (TORC1 Organized in Inhibited Domains) in the sample are composed of Kog1 (P38873), Lst8 (P41318) and Tor2 (P32600)
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / 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

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: TORC1 Organized in Inhibited Domains (TOROID) / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Strain: RL174-5b: MATa; TB50, KOG1::TAP-HIS3 tor1Delta::KanMX6
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM2-(N-morpholino)ethanesulfonic acid-sodium hydroxideMES-NaOH1
25 mM3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonateCHAPS1
3600 mMsodium chlorideNaClSodium chloride1
40.5 mMDithiothreitolDTT1
51 Phosphatase Inhibitor Mix1
61 1 tablet/50 mlComplete Complete Protease Inhibitor Cocktail (Roche)1
71 mMphenylmethylsulfonyl fluoridePMSFPMSF1
SpecimenConc.: 0.02 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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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: OTHER / Nominal magnification: 37000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 8 sec. / Electron dose: 20 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4901
Image scansMovie frames/image: 16 / Used frames/image: 2-16

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1EMAN2particle selectionEMAN2 e2helixboxer.py was used to manually select TOROID filaments.
4CTFFIND4CTF correction
5GctfCTF correction
8UCSF Chimeramodel fitting
9iMODFITmodel fitting
10NAMDmodel fitting
12PHENIXmodel refinement
13RELION2initial Euler assignment
14cryoSPARC3.01final Euler assignment
15cryoSPARC3.01classification
16cryoSPARC3.013D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 219455
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 218872
Details: Based on an initial helical reconstruction in RELION2.0, we performed signal subtraction to generate a set of 219,455 subtracted particles containing one isolated Torc1 assembly per segment. ...Details: Based on an initial helical reconstruction in RELION2.0, we performed signal subtraction to generate a set of 219,455 subtracted particles containing one isolated Torc1 assembly per segment. Next, we employed the localrec module in Scipion to crop and re-center the signal subtracted particles in a smaller box of 300 pixels, as well as assigning to each particle a refined defocus based on the helical geometry. Single-particle analysis of the subtracted particles using 3D refinement in RELION2.0, while imposing D1 symmetry, resulted in a map with a resolution of 4.5 Angstrom (FSC = 0.143). We then imported the particles from the RELION2.0 refinement in cryoSPARC 3.01. After 2D classification and class selection, a set of 213808 selected particles was used for Non-Uniform refinement in cryoSPARC 3.01, employing a dynamic mask, imposing D1 symmetry, using the option to keep particles from the same helix in the same half-set, and allowing high-order aberration estimation and correction, which resulted in a final map with a resolution of 3.87 Angstrom (FSC = 0.143).
Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Details: Initial homology models of Tor2 and Lst8 were generated using Phyre2, while a model of Kog1 was generated using ITasser. Homology models of Tor2, Lst8 and Kog1 were first manually placed in ...Details: Initial homology models of Tor2 and Lst8 were generated using Phyre2, while a model of Kog1 was generated using ITasser. Homology models of Tor2, Lst8 and Kog1 were first manually placed in the final 3D map followed by rigid-body fitting in Chimera. The rigid-body fitted models were subsequently subjected to a round of flexible fitting using Imodfit followed by automatic molecular dynamics flexible fitting using NAMDINATOR. The flexibly fitted structure was then refined using the Phenix software package.
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 121.52 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004104927
ELECTRON MICROSCOPYf_angle_d0.861142305
ELECTRON MICROSCOPYf_dihedral_angle_d5.40813747
ELECTRON MICROSCOPYf_chiral_restr0.05116205
ELECTRON MICROSCOPYf_plane_restr0.00618069

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