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Yorodumi- EMDB-10132: cryo-EM structure of mTORC1 bound to PRAS40-fused active RagA/C G... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-10132 | ||||||||||||
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Title | cryo-EM structure of mTORC1 bound to PRAS40-fused active RagA/C GTPases | ||||||||||||
Map data | CryoEM structure of RagA/C heterodimer GTPases in complex with mTORC1 | ||||||||||||
Sample |
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Keywords | small GTPases / mTORC1 activator / roadblock domain / GTPase domain / SIGNALING PROTEIN | ||||||||||||
Function / homology | Function and homology information Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / cellular response to leucine starvation ...Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / 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 / regulation of TORC1 signaling / negative regulation of lysosome organization / RNA polymerase III type 3 promoter sequence-specific DNA binding / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / TORC1 complex / protein localization to lysosome / calcineurin-NFAT signaling cascade / nucleus localization / cellular response to methionine / voluntary musculoskeletal movement / regulation of osteoclast differentiation / regulation of TOR signaling / positive regulation of odontoblast differentiation / TORC1 signaling / positive regulation of keratinocyte migration / cellular response to L-leucine / MTOR signalling / Amino acids regulate mTORC1 / cellular response to nutrient / regulation of autophagosome assembly / Energy dependent regulation of mTOR by LKB1-AMPK / energy reserve metabolic process / negative regulation of cell size / ruffle organization / protein localization to membrane / protein serine/threonine kinase inhibitor activity / positive regulation of osteoclast differentiation / cellular response to osmotic stress / negative regulation of TOR signaling / negative regulation of protein localization to nucleus / enzyme-substrate adaptor activity / anoikis / cardiac muscle cell development / AKT phosphorylates targets in the cytosol / positive regulation of transcription by RNA polymerase III / negative regulation of calcineurin-NFAT signaling cascade / regulation of myelination / negative regulation of macroautophagy / regulation of cell size / small GTPase-mediated signal transduction / positive regulation of G1/S transition of mitotic cell cycle / positive regulation of oligodendrocyte differentiation / Macroautophagy / positive regulation of actin filament polymerization / lysosome organization / positive regulation of myotube differentiation / protein kinase inhibitor activity / protein kinase activator activity / oligodendrocyte differentiation / behavioral response to pain / Constitutive Signaling by AKT1 E17K in Cancer / germ cell development / mTORC1-mediated signalling / CD28 dependent PI3K/Akt signaling / : / neurotrophin TRK receptor signaling pathway / social behavior / HSF1-dependent transactivation / neuronal action potential / TOR signaling / positive regulation of TOR signaling / response to amino acid / endomembrane system / cellular response to nutrient levels / positive regulation of translational initiation / regulation of macroautophagy / 'de novo' pyrimidine nucleobase biosynthetic process / positive regulation of lamellipodium assembly / positive regulation of epithelial to mesenchymal transition / positive regulation of lipid biosynthetic process / regulation of neuron apoptotic process / heart morphogenesis / response to nutrient / cardiac muscle contraction / regulation of cellular response to heat / protein-membrane adaptor activity / phagocytic vesicle / positive regulation of stress fiber assembly / negative regulation of TORC1 signaling / cytoskeleton organization / 14-3-3 protein binding / Regulation of PTEN gene transcription / tumor necrosis factor-mediated signaling pathway / positive regulation of endothelial cell proliferation / T cell costimulation / positive regulation of TORC1 signaling Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.5 Å | ||||||||||||
Authors | Anandapadamanaban M / Berndt A | ||||||||||||
Funding support | United Kingdom, 3 items
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Citation | Journal: Science / Year: 2019 Title: Architecture of human Rag GTPase heterodimers and their complex with mTORC1. Authors: Madhanagopal Anandapadamanaban / Glenn R Masson / Olga Perisic / Alex Berndt / Jonathan Kaufman / Chris M Johnson / Balaji Santhanam / Kacper B Rogala / David M Sabatini / Roger L Williams / Abstract: The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag ...The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagA/RagC nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_10132.map.gz | 150.2 MB | EMDB map data format | |
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Header (meta data) | emd-10132-v30.xml emd-10132.xml | 35.6 KB 35.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_10132_fsc.xml | 12.4 KB | Display | FSC data file |
Images | emd_10132.png | 263.6 KB | ||
Masks | emd_10132_msk_1.map | 163.6 MB | Mask map | |
Filedesc metadata | emd-10132.cif.gz | 11.1 KB | ||
Others | emd_10132_additional.map.gz emd_10132_half_map_1.map.gz emd_10132_half_map_2.map.gz | 151.6 MB 129 MB 129.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-10132 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10132 | HTTPS FTP |
-Validation report
Summary document | emd_10132_validation.pdf.gz | 919.3 KB | Display | EMDB validaton report |
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Full document | emd_10132_full_validation.pdf.gz | 918.9 KB | Display | |
Data in XML | emd_10132_validation.xml.gz | 19 KB | Display | |
Data in CIF | emd_10132_validation.cif.gz | 24.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10132 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10132 | HTTPS FTP |
-Related structure data
Related structure data | 6sb0MC 6s6aC 6s6dC 6sb2C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_10132.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | CryoEM structure of RagA/C heterodimer GTPases in complex with mTORC1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.43 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_10132_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: The experimental EM map of 3D reconstruction of mTORC1-RagA/C monomer
File | emd_10132_additional.map | ||||||||||||
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Annotation | The experimental EM map of 3D reconstruction of mTORC1-RagA/C monomer | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half1 map from 3D reconstruction RELION-3.0.6
File | emd_10132_half_map_1.map | ||||||||||||
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Annotation | Half1 map from 3D reconstruction RELION-3.0.6 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half2 map from 3D reconstruction RELION-3.0.6
File | emd_10132_half_map_2.map | ||||||||||||
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Annotation | Half2 map from 3D reconstruction RELION-3.0.6 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : cryoEM structure of mTORC1 bound to RagA/C complex
+Supramolecule #1: cryoEM structure of mTORC1 bound to RagA/C complex
+Supramolecule #2: mTORC1
+Supramolecule #3: RagA/C
+Macromolecule #1: mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-p...
+Macromolecule #2: Target of rapamycin complex subunit LST8
+Macromolecule #3: Ras-related GTP-binding protein A
+Macromolecule #4: Ras-related GTP-binding protein C
+Macromolecule #5: Regulatory-associated protein of mTOR
+Macromolecule #6: Proline-rich AKT1 substrate 1
+Macromolecule #7: GUANOSINE-5'-TRIPHOSPHATE
+Macromolecule #8: GUANOSINE-5'-DIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.05 mg/mL |
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Buffer | pH: 7 / Details: 50mM HEPES pH 7.0, 100mM NaCl, 2mM MgCl2, 1mM TCEP |
Grid | Model: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK III |
Details | mTORC1 (mTOR complex 1) is a dimer consists of three proteins: mTOR, mLST8 and RAPTOR. The interacting partner, PRAS40-fused-RagA/C (referred as RagA/C here) forms complex with mTORC1 for its activation. We solved the cryo-EM structure of mTORC1 bound to RagA/C. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-22 / Average exposure time: 1.8 sec. / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Details | Cryo-EM model of mTORC1-RagA/C was refined using REFMAC5 program in CCPEM package, with a composite map of the 3D reconstruction of mTORC1-RagA/C pseudo-monomer (as mentioned in Reconstruction section) of one protomer together with the generated map for the other second protomer of mTORC1-RagA/C. This second protomer of mTORC1-RagA/C map was generated by simply aligning the first 3D reconstructed pseudomonomer map onto the mTORC1 dimer consensus C2 map and then obtained the rotation-translation matrix with CHIMERA and then used Maputils program in CCP4i. From the resulting mTORC1-RagA/C dimer map, the model of mTORC1-RagA/C was built by using previously published structure of apo-mTORC1 (PDB ID 6BCX) and our crystal structure of RagA/C was fitted (PDB ID 6S6A, unreleased). The entire mTORC1-RagA/C final model was refined using REFMAC5 program using the restraints from the crystal structure of RagA/C and previously published mTORC1 structure. Side chains were removed before refinement, since these were not evident in the cryo-EM densities. Separate model refinements were performed against single half-maps, and the resulting models were compared with the other half-maps to confirm the absence of overfitting. | ||||||
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 283 | ||||||
Output model | PDB-6sb0: |