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

Basic information

• Entry: Database: EMDB / ID: EMD-0556
• Title: Cryo-EM Map of the active Ragulator-RagA-RagC Complex
• Sample: active RagA-RagC-Ragulator Complex

Function / homology

Function:

negative regulation of cell proliferation involved in kidney development / negative regulation of brown fat cell differentiation / regulation of cholesterol esterification / regulation of cholesterol import / regulation of cell-substrate junction organization / Gtr1-Gtr2 GTPase complex / regulation of TORC1 signaling / regulation of cholesterol efflux / Ragulator complex / EGO complex / protein localization to cell junction / regulation of Ras protein signal transduction / protein localization to lysosome / positive regulation of RNA polymerase II regulatory region sequence-specific DNA binding / negative regulation of TORC1 signaling / ATPase inhibitor activity / lysosome localization / negative regulation of ATP biosynthetic process / positive regulation of TORC1 signaling / regulation of pro-B cell differentiation / regulation of TOR signaling / regulation of histone acetylation / fibroblast migration / kinase activator activity / negative regulation of TOR signaling / endosome organization / modulation by virus of host process / lysosome organization / positive regulation of TOR signaling / positive regulation of interleukin-8 production => GO:0032757 / negative regulation of mitochondrion organization / negative regulation of Rho protein signal transduction / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / azurophil granule membrane / cell death / cell-cell junction assembly / negative regulation of cold-induced thermogenesis / negative regulation of protein localization to nucleus / regulation of cell size / positive regulation of transforming growth factor beta receptor signaling pathway / small GTPase mediated signal transduction / centriolar satellite / ficolin-1-rich granule membrane / tertiary granule membrane / TOR signaling / negative regulation of protein kinase B signaling / energy homeostasis / hemopoiesis / positive regulation of cell adhesion / regulation of receptor recycling / regulation of macroautophagy / specific granule membrane / late endosome membrane / cellular response to amino acid starvation / molecular adaptor activity / positive regulation of autophagy / guanyl-nucleotide exchange factor activity / cellular protein localization / activation of MAPKK activity / negative regulation of cell migration / response to amino acid / GTPase binding / cilium / regulation of autophagy / cellular response to starvation / RNA splicing / GTPase activator activity / negative regulation of autophagy / cellular response to amino acid stimulus / viral genome replication / regulation of cell growth / regulation of protein phosphorylation / mitotic spindle / negative regulation of ERK1 and ERK2 cascade / regulation of cytokinesis / cholesterol homeostasis / endosomal transport / response to virus / positive regulation of protein-containing complex assembly / phosphoprotein binding / GDP binding / positive regulation of protein localization to nucleus / positive regulation of GTPase activity / cell cycle arrest / negative regulation of cysteine-type endopeptidase activity involved in apoptotic process / negative regulation of cell growth / intrinsic apoptotic signaling pathway in response to DNA damage / lysosomal membrane / late endosome / positive regulation of MAPK cascade / chaperone binding / positive regulation of I-kappaB kinase/NF-kappaB signaling / positive regulation of peptidyl-serine phosphorylation / in utero embryonic development / lysosome / endosome membrane / positive regulation of NF-kappaB transcription factor activity / MAPK cascade / protein ubiquitination / transcription, DNA-templated

Domain/homology:

Ragulator complex protein LAMTOR3 / Folliculin-interacting protein, middle domain / Roadblock/LAMTOR2 domain / Gtr1/RagA G protein / Folliculin / Ragulator complex protein LAMTOR5 / Folliculin-interacting protein family / P-loop containing nucleoside triphosphate hydrolase / Folliculin-interacting protein, N-terminal domain / Folliculin-interacting protein, C-terminal domain / LAMTOR1/MEH1 / Folliculin, C-terminal / Ragulator complex protein LAMTOR4 / Folliculin/SMCR8, N-terminal / Folliculin/SMCR8, tripartite DENN domain / Tripartite DENN domain, FNIP1/2-type / Ragulator complex protein LAMTOR2-like / RagA/B / RagC/D

Component:

Late endosomal/lysosomal adaptor and MAPK and MTOR activator 5 / 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

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 8.1 Å
• Authors: Yokom AL / Fromm SA / Hurley JH

Funding support

United States, 1 items

Organization	Grant number	Country
National Institutes of Health/National Institute of General Medical Sciences	2R01GM111730-05	United States

• Citation: Journal: Science (New York, N.Y.) / 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) 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

Deposition	Feb 13, 2019	-
Header (metadata) release	Feb 20, 2019	-
Map release	Nov 6, 2019	-
Update	Dec 4, 2019	-
Current status	Dec 4, 2019	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_0556.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.149 Å

Density

Contour Level	By AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum	-0.8477015 - 1.839886
Average (Standard dev.)	0.0055222926 (±0.050337825)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 200 200 200 Spacing 200 200 200
Cell	A=B=C: 229.8 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.149	1.149	1.149
M x/y/z	200	200	200
origin x/y/z	0.000	0.000	0.000
length x/y/z	229.800	229.800	229.800
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	200	200	200
D min/max/mean	-0.848	1.840	0.006

Supplemental data

Sample components

Entire active RagA-RagC-Ragulator Complex

• Entire: Name: active RagA-RagC-Ragulator Complex; Details: RagA bound to GTP; RagC bound to XDP RagC has T90N and D181N mutations; Number of components: 1

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

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

Experimental details

Sample preparation

• Specimen: Specimen state: Particle / Method: cryo EM
• Sample solution: Specimen conc.: 0.5 mg/mL / pH: 7.4
• Vitrification: Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 90 %

Electron microscopy imaging

• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company
• Imaging: Microscope: FEI TITAN KRIOS
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 48 e/Ų / Illumination mode: OTHER
• Lens: Cs: 2.7 mm / Imaging mode: BRIGHT FIELD
• Specimen Holder: Model: FEI TITAN KRIOS AUTOGRID HOLDER
• Camera: Detector: GATAN K2 SUMMIT (4k x 4k)

Image processing

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