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- PDB-7uhy: Human GATOR2 complex -

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Basic information

Entry
Database: PDB / ID: 7uhy
TitleHuman GATOR2 complex
Components
  • (GATOR complex protein ...) x 3
  • (Unknown) x 2
  • Isoform B of Nucleoporin SEH1
  • Protein SEC13 homolog
KeywordsSIGNALING PROTEIN / mTOR / GATOR / lysosome / nutrient sensing / complex / zinc finger / ZnF / RING / C2 symmetry / octagon
Function / homology
Function and homology information


oligodendrocyte progenitor proliferation / GATOR2 complex / Seh1-associated complex / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / COPII-coated vesicle cargo loading / nuclear pore organization / nuclear pore outer ring / central nervous system myelin formation / COPII vesicle coat ...oligodendrocyte progenitor proliferation / GATOR2 complex / Seh1-associated complex / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / COPII-coated vesicle cargo loading / nuclear pore organization / nuclear pore outer ring / central nervous system myelin formation / COPII vesicle coat / Nuclear Pore Complex (NPC) Disassembly / Transport of Ribonucleoproteins into the Host Nucleus / Amino acids regulate mTORC1 / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / attachment of mitotic spindle microtubules to kinetochore / protein K6-linked ubiquitination / Transport of the SLBP independent Mature mRNA / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways / SUMOylation of SUMOylation proteins / protein-containing complex localization / Transport of Mature mRNA Derived from an Intronless Transcript / Rev-mediated nuclear export of HIV RNA / Nuclear import of Rev protein / SUMOylation of RNA binding proteins / NEP/NS2 Interacts with the Cellular Export Machinery / tRNA processing in the nucleus / Transport of Mature mRNA derived from an Intron-Containing Transcript / Postmitotic nuclear pore complex (NPC) reformation / COPII-mediated vesicle transport / nucleocytoplasmic transport / vacuolar membrane / Viral Messenger RNA Synthesis / mitotic metaphase chromosome alignment / SUMOylation of ubiquitinylation proteins / Vpr-mediated nuclear import of PICs / oligodendrocyte differentiation / positive regulation of macroautophagy / SUMOylation of DNA replication proteins / positive regulation of TOR signaling / Regulation of HSF1-mediated heat shock response / mRNA transport / cellular response to nutrient levels / nuclear pore / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / SUMOylation of DNA damage response and repair proteins / signaling adaptor activity / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / negative regulation of TORC1 signaling / MHC class II antigen presentation / Resolution of Sister Chromatid Cohesion / positive regulation of TORC1 signaling / cellular response to amino acid starvation / SUMOylation of chromatin organization proteins / HCMV Late Events / regulation of autophagy / RHO GTPases Activate Formins / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / Transcriptional regulation by small RNAs / intracellular protein transport / RING-type E3 ubiquitin transferase / ER to Golgi transport vesicle membrane / kinetochore / ISG15 antiviral mechanism / autophagy / HCMV Early Events / protein import into nucleus / Separation of Sister Chromatids / ubiquitin protein ligase activity / protein transport / cell junction / nuclear envelope / snRNP Assembly / defense response to Gram-positive bacterium / cell division / lysosomal membrane / intracellular membrane-bounded organelle / endoplasmic reticulum membrane / SARS-CoV-2 activates/modulates innate and adaptive immune responses / structural molecule activity / extracellular exosome / nucleoplasm / identical protein binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
WDR59, modified RING finger, H2 subclass (C3H3C2-type) / WD repeat protein mio, zinc-ribbon like domain / GATOR complex protein WDR24 / MIOS/Sea4 / : / Zinc-ribbon like family / MIOS, alpha-solenoid / : / : / RING/Ubox like zinc-binding domain ...WDR59, modified RING finger, H2 subclass (C3H3C2-type) / WD repeat protein mio, zinc-ribbon like domain / GATOR complex protein WDR24 / MIOS/Sea4 / : / Zinc-ribbon like family / MIOS, alpha-solenoid / : / : / RING/Ubox like zinc-binding domain / MIOS, WD40 repeat / RWD domain profile. / RWD / RWD domain / Sec13/Seh1 family / Ubiquitin-conjugating enzyme/RWD-like / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Protein SEC13 homolog / GATOR2 complex protein WDR59 / Nucleoporin SEH1 / GATOR2 complex protein WDR24 / GATOR2 complex protein MIOS
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsRogala, K.B. / Valenstein, M.L. / Lalgudi, P.V.
Funding support United States, 17items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)K99 CA255926 United States
Tuberous Sclerosis Association2018-F01 United States
Other privateCharles King Trust
Lustgarten Foundation United States
Department of Defense (DOD, United States)TS200035 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM007753 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)F30 CA228229 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)F31 CA180271 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31 GM121093 United States
Other privateKoch Institute Graduate Fellowship
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA103866 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA129105 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI047389 United States
Department of Defense (DOD, United States)W81XWH-21-1-0260 United States
Other privateLeo Foundation
American Cancer Society United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2022
Title: Structure of the nutrient-sensing hub GATOR2.
Authors: Max L Valenstein / Kacper B Rogala / Pranav V Lalgudi / Edward J Brignole / Xin Gu / Robert A Saxton / Lynne Chantranupong / Jonas Kolibius / Jan-Philipp Quast / David M Sabatini /
Abstract: Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients. Amino acids signal to mTORC1 ...Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 β-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through β-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes. The scaffold orients the WD40 β-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.
History
DepositionMar 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 27, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 3, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 20, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GATOR complex protein MIOS
B: GATOR complex protein MIOS
C: GATOR complex protein WDR24
D: GATOR complex protein WDR59
E: Isoform B of Nucleoporin SEH1
F: Isoform B of Nucleoporin SEH1
G: Isoform B of Nucleoporin SEH1
H: Protein SEC13 homolog
I: Unknown
J: Unknown
hetero molecules


Theoretical massNumber of molelcules
Total (without water)578,05926
Polymers577,01310
Non-polymers1,04716
Water00
1
A: GATOR complex protein MIOS
B: GATOR complex protein MIOS
C: GATOR complex protein WDR24
D: GATOR complex protein WDR59
E: Isoform B of Nucleoporin SEH1
F: Isoform B of Nucleoporin SEH1
G: Isoform B of Nucleoporin SEH1
H: Protein SEC13 homolog
I: Unknown
J: Unknown
hetero molecules

A: GATOR complex protein MIOS
B: GATOR complex protein MIOS
C: GATOR complex protein WDR24
D: GATOR complex protein WDR59
E: Isoform B of Nucleoporin SEH1
F: Isoform B of Nucleoporin SEH1
G: Isoform B of Nucleoporin SEH1
H: Protein SEC13 homolog
I: Unknown
J: Unknown
hetero molecules


  • complete point assembly
  • Evidence: gel filtration, Peak elution of the recombinant protein complex matches that of the endogenous GATOR2. Elution volume of 8.5 mL from a TSKgel G4000SWxl column corresponds to a large MDa- ...Evidence: gel filtration, Peak elution of the recombinant protein complex matches that of the endogenous GATOR2. Elution volume of 8.5 mL from a TSKgel G4000SWxl column corresponds to a large MDa-size particle., electron microscopy, Clear intact C2-symmetric particles, 30x20 nm in size.
  • 1.16 MDa, 20 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)1,156,11952
Polymers1,154,02620
Non-polymers2,09332
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation1
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C2 (2 fold cyclic))
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-1), (-1), (1)419.40482, 419.40482

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Components

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GATOR complex protein ... , 3 types, 4 molecules ABCD

#1: Protein GATOR complex protein MIOS / Missing oocyte meiosis regulator homolog


Mass: 100633.383 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MIOS / Cell line (production host): HEK-293T / Production host: Homo sapiens (human) / References: UniProt: Q9NXC5
#2: Protein GATOR complex protein WDR24 / WD repeat-containing protein 24


Mass: 88326.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR24, C16orf21, JFP7 / Cell line (production host): HEK-293T / Production host: Homo sapiens (human) / References: UniProt: Q96S15
#3: Protein GATOR complex protein WDR59 / WD repeat-containing protein 59


Mass: 109938.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR59, KIAA1923, FP977 / Cell line (production host): CRL-3216 / Production host: Homo sapiens (human) / Variant (production host): HEK-293T / References: UniProt: Q6PJI9

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Protein , 2 types, 4 molecules EFGH

#4: Protein Isoform B of Nucleoporin SEH1 / GATOR complex protein SEH1 / Nup107-160 subcomplex subunit SEH1 / SEC13-like protein


Mass: 46636.289 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SEH1L, SEC13L, SEH1 / Cell line (production host): HEK-293T / Production host: Homo sapiens (human) / References: UniProt: Q96EE3
#5: Protein Protein SEC13 homolog / GATOR complex protein SEC13 / SEC13-like protein 1 / SEC13-related protein


Mass: 35578.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SEC13, D3S1231E, SEC13A, SEC13L1, SEC13R / Cell line (production host): HEK-293T / Production host: Homo sapiens (human) / References: UniProt: P55735

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Protein/peptide , 2 types, 2 molecules IJ

#6: Protein/peptide Unknown


Mass: 1294.587 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK-293T / Production host: Homo sapiens (human)
#7: Protein/peptide Unknown


Mass: 698.854 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK-293T / Production host: Homo sapiens (human)

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Non-polymers , 1 types, 16 molecules

#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human GATOR2 / Type: COMPLEX
Details: Purified five-component GATOR2 complex via a Flag-tag on MIOS.
Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 1.1 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK-293T
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
2150 mMsodium chloride1
30.1 %CHAPS1
450 mML-arginine1
550 mML-glutamate1
62 mMmagnesium chloride1
72 mMdithiothreitol1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 95% pure, monodisperse protein complex. Partial loss of a few subunits observed. Contaminating CCT chaperonin present.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 45779 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording

Imaging-ID: 1 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

IDAverage exposure time (sec.)Electron dose (e/Å2)Num. of grids imagedNum. of real imagesDetails
12.44414103Movie stacks were recorded in super-resolution counting mode at the UMass Medical School's Cryo-EM Core Facility in Worcester, Massachusetts, USA. 30 frames per stack.
2447228792Movie stacks were recorded in super-resolution counting mode at MIT.nano in Cambridge, Massachusetts, USA. 30 frames per stack.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEM3.8.0image acquisitionUMass dataset
3EPU2.7.0image acquisitionMIT.nano dataset
5Gctf1.06CTF correction
8Coot0.9.7model fitting
9UCSF Chimera1.16model fitting
11PHENIX1.20.1model refinement
12RELION3initial Euler assignment
13RELION3.1final Euler assignment
14RELION3.1classification
15cryoSPARC3.2.03D reconstruction
Image processingDetails: Movie frames were weighted according to electron dose and particle movement. Please note that the two datasets collected at (1) UMass and (2) MIT.nano were eventually pixel-size-matched and ...Details: Movie frames were weighted according to electron dose and particle movement. Please note that the two datasets collected at (1) UMass and (2) MIT.nano were eventually pixel-size-matched and merged into a single dataset.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7237873
Details: The reported particle number is the sum of particles from two datasets, including parallel picking strategies. See details in the methods section of Valenstein and Rogala et al (2022).
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 784651 / Algorithm: FOURIER SPACE / Details: Duplicate particles were removed. / Num. of class averages: 5 / Symmetry type: POINT
Atomic model buildingB value: 182 / Protocol: OTHER / Space: REAL
Details: Most of the structure was built de novo in Coot. WD40 propellers were rebuilt after fitting either deposited PDB coordinates of homologous structures or predicted models from RoseTTAFold.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
14L9O

4l9o

B4L9O11043-2298
23JRP

3jrp

A3JRP23-1179
33F3F

3f3f

A3F3F31-346
43F3F

3f3f

C3F3F347-99
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00352976
ELECTRON MICROSCOPYf_angle_d0.41972474
ELECTRON MICROSCOPYf_dihedral_angle_d8.97916986
ELECTRON MICROSCOPYf_chiral_restr0.0418356
ELECTRON MICROSCOPYf_plane_restr0.0039382

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