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- EMDB-0556: Cryo-EM Map of the active Ragulator-RagA-RagC Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0556
TitleCryo-EM Map of the active Ragulator-RagA-RagC Complex
Map dataem-volume_P1
Sample
  • Complex: active RagA-RagC-Ragulator Complex
Function / homology
Function and homology information


negative regulation of cell proliferation involved in kidney development / negative regulation of mitochondrial DNA metabolic process / negative regulation of muscle tissue development / negative regulation of post-translational protein modification / cell proliferation involved in kidney development / : / regulation of cholesterol import / positive regulation of protein localization to lysosome / regulation of cell-substrate junction organization / Gtr1-Gtr2 GTPase complex ...negative regulation of cell proliferation involved in kidney development / negative regulation of mitochondrial DNA metabolic process / negative regulation of muscle tissue development / negative regulation of post-translational protein modification / cell proliferation involved in kidney development / : / regulation of cholesterol import / positive regulation of protein localization to lysosome / regulation of cell-substrate junction organization / Gtr1-Gtr2 GTPase complex / regulation of cholesterol efflux / negative regulation of brown fat cell differentiation / positive regulation of RNA polymerase II regulatory region sequence-specific DNA binding / negative regulation of cellular respiration / FNIP-folliculin RagC/D GAP / Ragulator complex / regulation of Ras protein signal transduction / protein localization to cell junction / : / regulation of TORC1 signaling / negative regulation of lysosome organization / negative regulation of ATP biosynthetic process / regulation of pro-B cell differentiation / protein localization to lysosome / TORC1 signaling / regulation of TOR signaling / endosome organization / ATPase inhibitor activity / MTOR signalling / Amino acids regulate mTORC1 / fibroblast migration / lysosome localization / Energy dependent regulation of mTOR by LKB1-AMPK / negative regulation of TOR signaling / kinase activator activity / protein localization to membrane / enzyme-substrate adaptor activity / enzyme inhibitor activity / cell-cell junction assembly / negative regulation of glycolytic process / negative regulation of cold-induced thermogenesis / negative regulation of protein localization to nucleus / negative regulation of Rho protein signal transduction / azurophil granule membrane / endosomal transport / Macroautophagy / regulation of cell size / lysosome organization / small GTPase-mediated signal transduction / RHOJ GTPase cycle / TOR signaling / RHOQ GTPase cycle / positive regulation of transforming growth factor beta receptor signaling pathway / mTORC1-mediated signalling / tertiary granule membrane / cellular response to nutrient levels / RHOH GTPase cycle / CDC42 GTPase cycle / hemopoiesis / ficolin-1-rich granule membrane / centriolar satellite / RHOG GTPase cycle / positive regulation of cell adhesion / negative regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of TOR signaling / regulation of receptor recycling / RAC3 GTPase cycle / RAC2 GTPase cycle / response to amino acid / specific granule membrane / positive regulation of autophagy / energy homeostasis / protein-membrane adaptor activity / positive regulation of intrinsic apoptotic signaling pathway / ERK1 and ERK2 cascade / positive regulation of TORC1 signaling / RAC1 GTPase cycle / tumor necrosis factor-mediated signaling pathway / cellular response to amino acid starvation / cellular response to starvation / GTPase activator activity / viral genome replication / intrinsic apoptotic signaling pathway / guanyl-nucleotide exchange factor activity / negative regulation of autophagy / cholesterol homeostasis / transforming growth factor beta receptor signaling pathway / negative regulation of cell migration / RNA splicing / phosphatidylinositol 3-kinase/protein kinase B signal transduction / regulation of cytokinesis / Regulation of PTEN gene transcription / epithelial cell proliferation / positive regulation of interleukin-8 production / regulation of cell growth / positive regulation of protein-containing complex assembly / cellular response to amino acid stimulus / TP53 Regulates Metabolic Genes / phosphoprotein binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
Similarity search - Function
Folliculin / Folliculin, DENN domain / Folliculin, DENN domain, C-terminal superfamily / Folliculin C-terminal domain / Folliculin-interacting protein family / Folliculin-interacting protein, N-terminal domain / Folliculin-interacting protein, middle domain / Folliculin-interacting protein, C-terminal domain / Folliculin-interacting protein N-terminus / Folliculin-interacting protein middle domain ...Folliculin / Folliculin, DENN domain / Folliculin, DENN domain, C-terminal superfamily / Folliculin C-terminal domain / Folliculin-interacting protein family / Folliculin-interacting protein, N-terminal domain / Folliculin-interacting protein, middle domain / Folliculin-interacting protein, C-terminal domain / Folliculin-interacting protein N-terminus / Folliculin-interacting protein middle domain / Folliculin-interacting protein C-terminus / Tripartite DENN domain, FNIP1/2-type / Tripartite DENN FNIP1/2-type domain profile. / Folliculin/SMCR8, longin domain / Folliculin/SMCR8, tripartite DENN domain / Vesicle coat protein involved in Golgi to plasma membrane transport / Tripartite DENN FLCN/SMCR8-type domain profile. / LAMTOR1/MEH1 / Late endosomal/lysosomal adaptor and MAPK and MTOR activator / Late endosomal/lysosomal adaptor and MAPK and MTOR activator / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR3 / Ragulator complex protein LAMTOR5 / RagA/B / Mitogen-activated protein kinase kinase 1 interacting / Ragulator complex protein LAMTOR5 / Mitogen-activated protein kinase kinase 1 interacting / Gtr1/RagA G protein / RagC/D / Gtr1/RagA G protein conserved region / Ragulator complex protein LAMTOR2-like / Roadblock/LAMTOR2 domain / Roadblock/LC7 domain / Roadblock/LC7 domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Ragulator complex protein LAMTOR5 / Ragulator complex protein LAMTOR5 / Ragulator complex protein LAMTOR4 / Ragulator complex protein LAMTOR1 / Ras-related GTP-binding protein A / Folliculin / Ras-related GTP-binding protein C / Folliculin-interacting protein 2 / Ragulator complex protein LAMTOR3 / Ragulator complex protein LAMTOR2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsYokom AL / Fromm SA / Hurley JH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences2R01GM111730-05 United States
CitationJournal: Science / Year: 2019
Title: Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex.
Authors: Rosalie E Lawrence / Simon A Fromm / Yangxue Fu / Adam L Yokom / Do Jin Kim / Ashley M Thelen / Lindsey N Young / Chun-Yan Lim / Avi J Samelson / James H Hurley / Roberto Zoncu /
Abstract: The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) ...The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagA:RagC GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo-electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.
History
DepositionFeb 13, 2019-
Header (metadata) releaseFeb 20, 2019-
Map releaseNov 6, 2019-
UpdateDec 4, 2019-
Current statusDec 4, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0556.png

Downloads & links

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Map

FileDownload / File: emd_0556.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationem-volume_P1
Voxel sizeX=Y=Z: 1.149 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.8477015 - 1.839886
Average (Standard dev.)0.0055222926 (±0.050337825)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 229.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1491.1491.149
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z229.800229.800229.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.8481.8400.006

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Supplemental data

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Sample components

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Entire : active RagA-RagC-Ragulator Complex

EntireName: active RagA-RagC-Ragulator Complex
Components
  • Complex: active RagA-RagC-Ragulator Complex

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Supramolecule #1: active RagA-RagC-Ragulator Complex

SupramoleculeName: active RagA-RagC-Ragulator Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Details: RagA bound to GTP; RagC bound to XDP RagC has T90N and D181N mutations
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: Sf9
Molecular weightTheoretical: 150 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
25.0 mMHEPES
130.0 mMsodium chlorideNaClSodium chloride
2.5 mMmagnesium chlorideMgCl2
0.5 mMTCEP
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 8.0 sec. / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Startup modelType of model: NONE / Details: cryoSPARC v2 ab-initio
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 75996

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