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Open data
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Basic information
Entry | Database: PDB / ID: 6sb2 | ||||||||||||
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Title | cryo-EM structure of mTORC1 bound to active RagA/C GTPases | ||||||||||||
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![]() | SIGNALING PROTEIN / small GTPases / mTORC1 activator / roadblock domain / GTPase domain | ||||||||||||
Function / homology | ![]() 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 protein localization to nucleus / enzyme-substrate adaptor activity / anoikis / cardiac muscle cell development / 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 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 / : / 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 / 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 / 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 / cellular response to starvation / phosphorylation / positive regulation of glycolytic process / cellular response to amino acid starvation / protein serine/threonine kinase activator activity / response to nutrient levels Similarity search - Function | ||||||||||||
Biological species | ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.2 Å | ||||||||||||
![]() | Anandapadamanaban, M. / Berndt, A. / Masson, G.R. / Perisic, O. / Williams, R.L. | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 1.2 MB | Display | ![]() |
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PDB format | ![]() | 828.9 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 10133MC ![]() 6s6aC ![]() 6s6dC ![]() 6sb0C C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 3 types, 6 molecules ABEHYN
#1: Protein | Mass: 287235.188 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() References: UniProt: P42345, non-specific serine/threonine protein kinase #2: Protein | Mass: 35910.090 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() #5: Protein | Mass: 149200.016 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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-Ras-related GTP-binding protein ... , 2 types, 4 molecules CIDJ
#3: Protein | Mass: 36600.195 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #4: Protein | Mass: 44284.832 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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-Non-polymers , 2 types, 4 molecules ![](data/chem/img/GTP.gif)
![](data/chem/img/GDP.gif)
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#6: Chemical | #7: Chemical | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Molecular weight | Value: 1.09 MDa / Experimental value: YES | ||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7 Details: 100mM Tris-HCl pH7.0, 260mM NaCl, 5mM MgCl2, 1mM TCEP | ||||||||||||||||||||||||
Specimen | Conc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: mTORC1 (mTOR complex 1) is a dimer consists of three proteins: mTOR, mLST8 and RAPTOR. The small GTPases, RagA/C in its active form bind to mTORC1 for activation. We solved the cryo-EM ...Details: mTORC1 (mTOR complex 1) is a dimer consists of three proteins: mTOR, mLST8 and RAPTOR. The small GTPases, RagA/C in its active form bind to mTORC1 for activation. We solved the cryo-EM structure of mTORC1 bound to RagA/C. | ||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 1.8 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
Image scans | Movie frames/image: 22 / Used frames/image: 1-22 |
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Processing
EM software |
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Image processing | Details: The selected images were processed using MotionCor2 within the RELION-3.0.6 package. | ||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 169971 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51902 / Algorithm: FOURIER SPACE Details: For the final reconstruction of mTORC1-RagA/C structure we used a strategy taking advantage of the relion particle symmetry expand program, and duplicated the C2-refined particles and ...Details: For the final reconstruction of mTORC1-RagA/C structure we used a strategy taking advantage of the relion particle symmetry expand program, and duplicated the C2-refined particles and applied the appropriate rotation and translation to generate a set of monomers. We performed mTORC1-RagA/C 'pseudo-monomer' focussed classification with signal subtraction and obtained a reconstruction of 6.2 A resolution map. This cryo-EM density corresponded to the mTORC1-RagA/C pseudomonomer, where the previously published structure for apo-mTORC1 (PDB ID 6BCX) and our high-resolution crystal structure of RagA/C (6S6A) were fitted with great confidence from our experimental analysis including Pulldown assays, mutational at per-residue level in the binding interface and HDX-Mass Spectrometry. Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 315 / Protocol: RIGID BODY FIT / Space: REAL 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 ...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. | ||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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