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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 data
Sampleactive RagA-RagC-Ragulator Complex
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
  • Download
  • 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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 200 pix.
= 229.8 Å
1.15 Å/pix.
x 200 pix.
= 229.8 Å
1.15 Å/pix.
x 200 pix.
= 229.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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
Details: RagA bound to GTP; RagC bound to XDP RagC has T90N and D181N mutations
Number of components: 1

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Component #1: protein, active RagA-RagC-Ragulator Complex

ProteinName: active RagA-RagC-Ragulator Complex
Details: RagA bound to GTP; RagC bound to XDP RagC has T90N and D181N mutations
Recombinant expression: No
MassTheoretical: 150 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Strain: Sf9

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/mL / pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 48 e/Å2 / Illumination mode: OTHER
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 75996
3D reconstructionSoftware: cryoSPARC / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF

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