+Open data
-Basic information
Entry | Database: PDB / ID: 7owg | ||||||||||||
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Title | human DEPTOR in a complex with mutant human mTORC1 A1459P | ||||||||||||
Components |
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Keywords | SIGNALING PROTEIN / kinase / PIKK / mTOR / cancer-associated mutation / DEPTOR / partial inhibitor / cancer / PDZ / non-canonical PDZ binding | ||||||||||||
Function / homology | Function and homology information negative regulation of TORC2 signaling / regulation of extrinsic apoptotic signaling pathway / RNA polymerase III type 2 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of pentose-phosphate shunt / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / cellular response to leucine starvation ...negative regulation of TORC2 signaling / regulation of extrinsic apoptotic signaling pathway / RNA polymerase III type 2 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of pentose-phosphate shunt / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / cellular response to leucine starvation / TFIIIC-class transcription factor complex binding / TORC2 complex / regulation of membrane permeability / heart valve morphogenesis / negative regulation of lysosome organization / RNA polymerase III type 3 promoter sequence-specific DNA binding / TORC1 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / calcineurin-NFAT signaling cascade / regulation of autophagosome assembly / TORC1 signaling / positive regulation of odontoblast differentiation / voluntary musculoskeletal movement / regulation of osteoclast differentiation / positive regulation of keratinocyte migration / cellular response to L-leucine / MTOR signalling / Amino acids regulate mTORC1 / cellular response to nutrient / energy reserve metabolic process / Energy dependent regulation of mTOR by LKB1-AMPK / phosphatidic acid binding / nucleus localization / negative regulation of TOR signaling / ruffle organization / protein serine/threonine kinase inhibitor activity / negative regulation of cell size / positive regulation of osteoclast differentiation / cellular response to osmotic stress / enzyme-substrate adaptor activity / anoikis / cardiac muscle cell development / negative regulation of protein localization to nucleus / positive regulation of transcription by RNA polymerase III / regulation of myelination / negative regulation of calcineurin-NFAT signaling cascade / Macroautophagy / regulation of cell size / negative regulation of macroautophagy / lysosome organization / positive regulation of oligodendrocyte differentiation / positive regulation of actin filament polymerization / protein kinase inhibitor activity / protein kinase activator activity / positive regulation of myotube differentiation / behavioral response to pain / TOR signaling / oligodendrocyte differentiation / mTORC1-mediated signalling / Constitutive Signaling by AKT1 E17K in Cancer / germ cell development / social behavior / cellular response to nutrient levels / CD28 dependent PI3K/Akt signaling / positive regulation of translational initiation / positive regulation of phosphoprotein phosphatase activity / neuronal action potential / HSF1-dependent transactivation / positive regulation of TOR signaling / positive regulation of G1/S transition of mitotic cell cycle / regulation of macroautophagy / positive regulation of epithelial to mesenchymal transition / endomembrane system / 'de novo' pyrimidine nucleobase biosynthetic process / response to amino acid / positive regulation of lamellipodium assembly / phagocytic vesicle / positive regulation of autophagy / positive regulation of lipid biosynthetic process / regulation of cellular response to heat / heart morphogenesis / cardiac muscle contraction / negative regulation of TORC1 signaling / positive regulation of stress fiber assembly / cytoskeleton organization / cellular response to amino acid starvation / T cell costimulation / positive regulation of endothelial cell proliferation / cellular response to starvation / protein serine/threonine kinase activator activity / positive regulation of glycolytic process / response to nutrient levels / negative regulation of autophagy / response to nutrient / post-embryonic development / 14-3-3 protein binding / positive regulation of translation / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / Regulation of PTEN gene transcription Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å | ||||||||||||
Authors | Heimhalt, M. / Berndt, A. / Wagstaff, J. / Anandapadamanaban, M. / Perisic, O. / Maslen, S. / McLaughlin, S. / Yu, W.-H. / Masson, G.R. / Boland, A. ...Heimhalt, M. / Berndt, A. / Wagstaff, J. / Anandapadamanaban, M. / Perisic, O. / Maslen, S. / McLaughlin, S. / Yu, W.-H. / Masson, G.R. / Boland, A. / Ni, X. / Yamashita, K. / Murshudov, G.N. / Skehel, M. / Freund, S.M. / Williams, R.L. | ||||||||||||
Funding support | United Kingdom, 3items
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Citation | Journal: Elife / Year: 2021 Title: Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace. Authors: Maren Heimhalt / Alex Berndt / Jane Wagstaff / Madhanagopal Anandapadamanaban / Olga Perisic / Sarah Maslen / Stephen McLaughlin / Conny Wing-Heng Yu / Glenn R Masson / Andreas Boland / ...Authors: Maren Heimhalt / Alex Berndt / Jane Wagstaff / Madhanagopal Anandapadamanaban / Olga Perisic / Sarah Maslen / Stephen McLaughlin / Conny Wing-Heng Yu / Glenn R Masson / Andreas Boland / Xiaodan Ni / Keitaro Yamashita / Garib N Murshudov / Mark Skehel / Stefan M Freund / Roger L Williams / Abstract: The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted ...The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7owg.cif.gz | 1.2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7owg.ent.gz | 1 MB | Display | PDB format |
PDBx/mmJSON format | 7owg.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ow/7owg ftp://data.pdbj.org/pub/pdb/validation_reports/ow/7owg | HTTPS FTP |
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-Related structure data
Related structure data | 13097MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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Noncrystallographic symmetry (NCS) | NCS oper:
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-Components
#1: Protein | Mass: 287570.312 Da / Num. of mol.: 1 / Mutation: A1459P Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MTOR, FRAP, FRAP1, FRAP2, RAFT1, RAPT1, mMTOR / Plasmid: pOPL146 / Details (production host): mTOR in pCAG vector / Cell line (production host): Expi293F / Production host: Homo sapiens (human) References: UniProt: P42345, non-specific serine/threonine protein kinase |
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#2: Protein | Mass: 35910.090 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MLST8, GBL, LST8 / Plasmid: pOPL95 / Details (production host): human LST8 cloned in pCAG / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q9BVC4 |
#3: Protein | Mass: 46365.832 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: DEPTOR, DEPDC6 / Plasmid: pOPL138 Details (production host): GST(tev)-Human_DEPTOR_1-409 (S204,N389 natural variant) Production host: Escherichia coli (E. coli) / Strain (production host): LOBSTR / References: UniProt: Q8TB45 |
#4: Protein | Mass: 149200.016 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RPTOR, KIAA1303, RAPTOR / Plasmid: pOPL119 / Details (production host): Human_Raptor nontagged, in pCAG / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q8N122 |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
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Molecular weight |
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Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 Details: 50 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM TCEP, 1 mM MgCl2, 500 uM AMP-PNP | ||||||||||||||||||||||||||||
Specimen | Conc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Purified mutant mTORC1 A1459P and DEPTOR were preincubated in the presence of MgCl2 and AMP-PNP for 20 min and used for cryo-EM grid preparation. | ||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 287 K / Details: blotting time of 2 s and a force of -15. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: -3000 nm / Nominal defocus min: -1400 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 100 K |
Image recording | Average exposure time: 2.5 sec. / Electron dose: 56 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4759 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Width: 6000 / Height: 4000 |
-Processing
Software | Name: REFMAC / Version: 5.8.0272 / Classification: refinement / Contact author: Garib N. Murshudov / Contact author email: garib[at]mrc-lmb.cam.ac.uk / Date: Feb 9, 2020 Description: (un)restrained refinement or idealisation of macromolecular structures | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 376784 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 500000 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 145.7 / Protocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: Correlation | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6BCX Pdb chain-ID: B / Pdb chain residue range: 1-2549 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 4.7→299.244 Å / Cor.coef. Fo:Fc: 0.974 / WRfactor Rwork: 0.342 / SU B: 108.861 / SU ML: 1.072 / Average fsc overall: 0.7062 / Average fsc work: 0.7062 / ESU R: 0.804 Details: Hydrogens have been added in their riding positions
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Solvent computation | Solvent model: BABINET MODEL | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 122.054 Å2 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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