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- PDB-9ce3: Structure of the TSC:WIPI3 lysosomal recruitment complex -

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Basic information

Entry
Database: PDB / ID: 9ce3
TitleStructure of the TSC:WIPI3 lysosomal recruitment complex
Components
  • Hamartin
  • Isoform 4 of Tuberin
  • TBC1 domain family member 7
  • Unknown fragment
  • WD repeat domain phosphoinositide-interacting protein 3
KeywordsONCOPROTEIN / Tuberous sclerosis complex / tumour suppressor / GTPase-activating proteins (GAP) / TSC-mTORC pathway
Function / homology
Function and homology information


memory T cell differentiation / TSC1-TSC2 complex binding / TSC1-TSC2 complex / Inhibition of TSC complex formation by PKB / regulation of insulin receptor signaling pathway / cellular response to decreased oxygen levels / glycophagy / nucleophagy / negative regulation of cilium assembly / regulation of cell-matrix adhesion ...memory T cell differentiation / TSC1-TSC2 complex binding / TSC1-TSC2 complex / Inhibition of TSC complex formation by PKB / regulation of insulin receptor signaling pathway / cellular response to decreased oxygen levels / glycophagy / nucleophagy / negative regulation of cilium assembly / regulation of cell-matrix adhesion / protein localization to phagophore assembly site / phagophore assembly site membrane / negative regulation of ATP-dependent activity / response to growth factor / cardiac muscle cell differentiation / activation of GTPase activity / Energy dependent regulation of mTOR by LKB1-AMPK / ATPase inhibitor activity / pexophagy / autophagy of mitochondrion / phosphatidylinositol-3-phosphate binding / cell projection organization / regulation of stress fiber assembly / negative regulation of cell size / phagophore assembly site / negative regulation of TOR signaling / phosphatidylinositol-3,5-bisphosphate binding / anoikis / regulation of small GTPase mediated signal transduction / TBC/RABGAPs / AKT phosphorylates targets in the cytosol / protein folding chaperone complex / negative regulation of macroautophagy / Macroautophagy / positive chemotaxis / negative regulation of mitophagy / D-glucose import / Constitutive Signaling by AKT1 E17K in Cancer / negative regulation of Wnt signaling pathway / regulation of endocytosis / associative learning / positive regulation of macroautophagy / positive regulation of GTPase activity / autophagosome assembly / positive regulation of focal adhesion assembly / phosphatase binding / vesicle-mediated transport / negative regulation of TORC1 signaling / lipid droplet / myelination / Hsp70 protein binding / protein folding chaperone / negative regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / negative regulation of insulin receptor signaling pathway / GTPase activator activity / cellular response to starvation / cell-matrix adhesion / positive regulation of protein ubiquitination / adult locomotory behavior / TP53 Regulates Metabolic Genes / neural tube closure / hippocampus development / kidney development / Hsp90 protein binding / response to insulin / synapse organization / cerebral cortex development / potassium ion transport / small GTPase binding / endocytosis / protein import into nucleus / intracellular protein localization / lamellipodium / protein-folding chaperone binding / heart development / cytoplasmic vesicle / cell cortex / protein-macromolecule adaptor activity / adaptive immune response / cell population proliferation / lysosome / regulation of cell cycle / postsynaptic density / protein stabilization / ciliary basal body / negative regulation of cell population proliferation / lysosomal membrane / perinuclear region of cytoplasm / Golgi apparatus / protein homodimerization activity / protein-containing complex / nucleus / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Tuberin / Tuberin-type domain / Tuberin, N-terminal / Tuberin / Domain of unknown function (DUF3384) / TBC1 domain family member 7 / TBC1 domain family member 7, domain 2 / Hamartin / Hamartin protein / Tuberin/Ral GTPase-activating protein subunit alpha ...Tuberin / Tuberin-type domain / Tuberin, N-terminal / Tuberin / Domain of unknown function (DUF3384) / TBC1 domain family member 7 / TBC1 domain family member 7, domain 2 / Hamartin / Hamartin protein / Tuberin/Ral GTPase-activating protein subunit alpha / Rap/Ran-GAP domain / Rap/Ran-GAP superfamily / Rap/ran-GAP / Rap GTPase activating proteins domain profile. / : / PROPPIN / Rab-GTPase-TBC domain / Rab-GTPase-TBC domain superfamily / Rab-GTPase-TBC domain / TBC/rab GAP domain profile. / Armadillo-like helical / Armadillo-type fold / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Tuberin / WD repeat domain phosphoinositide-interacting protein 3 / Hamartin / TBC1 domain family member 7
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBayly-Jones, C. / Lupton, C.J. / D'Andrea, L. / Ellisdon, A.M.
Funding support United States, Australia, 3items
OrganizationGrant numberCountry
Department of Defense (DOD, United States)W81XWH-19-1-0182 United States
Australian Research Council (ARC)DE240100992 Australia
Australian Research Council (ARC)LE170100016 Australia
CitationJournal: Sci Adv / Year: 2024
Title: Structure of the human TSC:WIPI3 lysosomal recruitment complex.
Authors: Charles Bayly-Jones / Christopher J Lupton / Laura D'Andrea / Yong-Gang Chang / Gareth D Jones / Joel R Steele / Hari Venugopal / Ralf B Schittenhelm / Michelle L Halls / Andrew M Ellisdon /
Abstract: Tuberous sclerosis complex (TSC) is targeted to the lysosomal membrane, where it hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, inhibiting mechanistic target of ...Tuberous sclerosis complex (TSC) is targeted to the lysosomal membrane, where it hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, inhibiting mechanistic target of rapamycin complex 1 (mTORC1). Loss-of-function mutations in TSC cause TSC disease, marked by excessive tumor growth. Here, we overcome a high degree of continuous conformational heterogeneity to determine the 2.8-Å cryo-electron microscopy (cryo-EM) structure of the complete human TSC in complex with the lysosomal recruitment factor WD repeat domain phosphoinositide-interacting protein 3 (WIPI3). We discover a previously undetected amino-terminal TSC1 HEAT repeat dimer that clamps onto a single TSC wing and forms a phosphatidylinositol phosphate (PIP)-binding pocket, which specifically binds monophosphorylated PIPs. These structural advances provide a model by which WIPI3 and PIP-signaling networks coordinate to recruit TSC to the lysosomal membrane to inhibit mTORC1. The high-resolution TSC structure reveals previously unrecognized mutational hotspots and uncovers crucial insights into the mechanisms of TSC dysregulation in disease.
History
DepositionJun 26, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Isoform 4 of Tuberin
B: Isoform 4 of Tuberin
C: Hamartin
D: Hamartin
E: TBC1 domain family member 7
F: WD repeat domain phosphoinositide-interacting protein 3
G: Unknown fragment
H: Unknown fragment


Theoretical massNumber of molelcules
Total (without water)736,9998
Polymers736,9998
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Mass photometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Isoform 4 of Tuberin / Tuberous sclerosis 2 protein


Mass: 199339.000 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSC2, TSC4 / Production host: Homo sapiens (human) / References: UniProt: P49815
#2: Protein Hamartin / Tuberous sclerosis 1 protein


Mass: 133001.609 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSC1, KIAA0243, TSC / Plasmid: pRK7 / Cell line (production host): Expi HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q92574
#3: Protein TBC1 domain family member 7 / Cell migration-inducing protein 23


Mass: 35016.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TBC1D7, TBC7, HSPC239 / Production host: Homo sapiens (human) / References: UniProt: Q9P0N9
#4: Protein WD repeat domain phosphoinositide-interacting protein 3 / WIPI-3 / WD repeat-containing protein 45-like / WDR45-like protein / WD repeat-containing protein ...WIPI-3 / WD repeat-containing protein 45-like / WDR45-like protein / WD repeat-containing protein 45B / WIPI49-like protein


Mass: 35222.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR45B, WDR45L, WIPI3 / Production host: Homo sapiens (human) / References: UniProt: Q5MNZ6
#5: Protein/peptide Unknown fragment


Mass: 1039.273 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
Has protein modificationN

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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: TSC:WIPI3 lysosomal recruitment complex (composite map)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.733 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi HEK293
Buffer solutionpH: 7.6 / Details: 20 mM HEPES (pH 7.6), 250 mM NaCl, 2 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acidHEPES1
2250 mMsodium chlorideNaCl1
32 mMthreo-1,4-Dimercapto-2,3-butanediolDTT1
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50.01 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.71 sec. / Electron dose: 46.54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12807

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.0.1particle selection
2Topazparticle selection
3crYOLOparticle selection
4EPU2.12.1.2782image acquisition
6CTFFINDCTF correction
9ISOLDEmodel fitting
11cryoSPARC4.0.1initial Euler assignment
12cryoSPARC4.0.1final Euler assignment
13RELION4final Euler assignment
14RELION4classification
16PHENIXmodel refinement
17Cootmodel refinement
18ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1176292 / Details: Template picking, blob picking, TOPAZ, crYOLO
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 200000 / Algorithm: FOURIER SPACE
Details: The global resolution of this composite is estimated based on the voxel average of focused reconstructions.
Symmetry type: POINT
Atomic model buildingB value: 96.4 / Protocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDSource nameType
1P49815A1AlphaFoldin silico model
2Q92574C2AlphaFoldin silico model
35EJC

5ejc

5EJC3PDBexperimental model
49C9I

9c9i

F9C9IF4PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00535798
ELECTRON MICROSCOPYf_angle_d0.66448451
ELECTRON MICROSCOPYf_dihedral_angle_d12.73813452
ELECTRON MICROSCOPYf_chiral_restr0.0445550
ELECTRON MICROSCOPYf_plane_restr0.0066178

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