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- PDB-8adl: Cryo-EM structure of the SEA complex -

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Basic information

Entry
Database: PDB / ID: 8adl
TitleCryo-EM structure of the SEA complex
Components
  • (Nitrogen permease regulator ...) x 2
  • Maintenance of telomere capping protein 5
  • Nucleoporin SEH1
  • Protein transport protein SEC13
  • Restriction of telomere capping protein 1
  • SEH-associated protein 4
  • Vacuolar membrane-associated protein IML1
KeywordsSIGNALING PROTEIN / GTPase activating protein / coatomer / TOR signaling
Function / homology
Function and homology information


GATOR1 complex / pseudohyphal growth / GATOR2 complex / urea transport / negative regulation of small GTPase mediated signal transduction / positive regulation of ER to Golgi vesicle-mediated transport / Seh1-associated complex / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / nuclear pore localization ...GATOR1 complex / pseudohyphal growth / GATOR2 complex / urea transport / negative regulation of small GTPase mediated signal transduction / positive regulation of ER to Golgi vesicle-mediated transport / Seh1-associated complex / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / nuclear pore localization / proline transport / COPII-mediated vesicle transport / regulation of TORC1 signaling / nuclear pore outer ring / positive regulation of protein exit from endoplasmic reticulum / Transport of Mature mRNA derived from an Intron-Containing Transcript / COPII vesicle coat / Regulation of HSF1-mediated heat shock response / regulation of autophagosome assembly / SUMOylation of SUMOylation proteins / fungal-type vacuole / structural constituent of nuclear pore / fungal-type vacuole membrane / SUMOylation of RNA binding proteins / negative regulation of TOR signaling / cellular response to nitrogen starvation / SUMOylation of chromatin organization proteins / vacuolar membrane / nucleocytoplasmic transport / positive regulation of macroautophagy / positive regulation of TOR signaling / mRNA transport / nuclear pore / negative regulation of TORC1 signaling / ERAD pathway / signaling adaptor activity / positive regulation of autophagy / positive regulation of TORC1 signaling / GTPase activator activity / cellular response to amino acid starvation / cell periphery / meiotic cell cycle / protein import into nucleus / nuclear envelope / protein transport / cellular response to oxidative stress / nuclear membrane / intracellular signal transduction / response to xenobiotic stimulus / endoplasmic reticulum membrane / positive regulation of DNA-templated transcription / structural molecule activity / endoplasmic reticulum / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
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 / Nitrogen permease regulator 3 ...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 / Nitrogen permease regulator 3 / Nitrogen permease regulator 2 / Vacuolar membrane-associated protein Iml1 / DEPDC5 protein, C-terminal / : / : / Nitrogen Permease regulator of amino acid transport activity 3 / Nitrogen permease regulator 2 / Vacuolar membrane-associated protein Iml1, N-terminal domain / DEPDC5 protein C-terminal region / GATOR1 complex protein NPRL3, C-terminal HTH / RWD domain / RWD domain profile. / RWD / RWD domain / Sec13/Seh1 family / Domain found in Dishevelled, Egl-10, and Pleckstrin (DEP) / DEP domain profile. / Domain found in Dishevelled, Egl-10, and Pleckstrin / DEP domain / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / Zinc finger RING-type profile. / Zinc finger, RING-type / WD domain, G-beta repeat / Zinc finger, RING/FYVE/PHD-type / Winged helix DNA-binding domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / Winged helix-like DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Mainly Beta
Similarity search - Domain/homology
SEH-associated protein 4 / Nitrogen permease regulator 3 / Nitrogen permease regulator 2 / Vacuolar membrane-associated protein IML1 / Nucleoporin SEH1 / Maintenance of telomere capping protein 5 / Protein transport protein SEC13 / Restriction of telomere capping protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsTafur, L. / Loewith, R.
Funding supportEuropean Union, 2items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)ALTF 79-2019European Union
European Research Council (ERC)TENDOEuropean Union
CitationJournal: Nature / Year: 2022
Title: Cryo-EM structure of the SEA complex.
Authors: Lucas Tafur / Kerstin Hinterndorfer / Caroline Gabus / Chiara Lamanna / Ariane Bergmann / Yashar Sadian / Farzad Hamdi / Fotis L Kyrilis / Panagiotis L Kastritis / Robbie Loewith /
Abstract: The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in ...The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid and glucose signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.
History
DepositionJul 8, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 16, 2022Group: Database references / Category: citation_author / Item: _citation_author.identifier_ORCID
Revision 1.3Nov 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4May 10, 2023Group: Database references / Refinement description / Structure summary
Category: entity / entity_name_com ...entity / entity_name_com / pdbx_initial_refinement_model / struct_ref / struct_ref_seq
Item: _entity.pdbx_description / _struct_ref.db_code ..._entity.pdbx_description / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession
Revision 1.5Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Maintenance of telomere capping protein 5
E: Nucleoporin SEH1
G: SEH-associated protein 4
H: Protein transport protein SEC13
A: Restriction of telomere capping protein 1
B: SEH-associated protein 4
D: Nucleoporin SEH1
F: Nucleoporin SEH1
Q: Maintenance of telomere capping protein 5
M: Nucleoporin SEH1
O: SEH-associated protein 4
P: Protein transport protein SEC13
I: Restriction of telomere capping protein 1
J: SEH-associated protein 4
L: Nucleoporin SEH1
N: Nucleoporin SEH1
U: Nitrogen permease regulator 3
W: Vacuolar membrane-associated protein IML1
S: Nitrogen permease regulator 2
V: Nitrogen permease regulator 3
X: Vacuolar membrane-associated protein IML1
T: Nitrogen permease regulator 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,099,96450
Polymers2,098,13222
Non-polymers1,83128
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area140750 Å2
ΔGint-720 kcal/mol
Surface area502310 Å2
MethodPISA

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Components

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Protein , 6 types, 18 molecules CQEDFMLNGBOJHPAIWX

#1: Protein Maintenance of telomere capping protein 5 / SEH-associated protein 3


Mass: 131104.062 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03897
#2: Protein
Nucleoporin SEH1 / Nuclear pore protein SEH1 / SEC13 homolog 1


Mass: 39170.758 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53011
#3: Protein
SEH-associated protein 4


Mass: 117775.750 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38164
#4: Protein Protein transport protein SEC13


Mass: 33082.965 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04491
#5: Protein Restriction of telomere capping protein 1 / SEH-associated protein 2


Mass: 149533.297 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08281
#7: Protein Vacuolar membrane-associated protein IML1 / Increased minichromosome loss protein 1 / SEH-associated protein 1


Mass: 182203.359 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P47170

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Nitrogen permease regulator ... , 2 types, 4 molecules UVST

#6: Protein Nitrogen permease regulator 3 / Required for meiotic nuclear division protein 11


Mass: 130141.094 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38742
#8: Protein Nitrogen permease regulator 2


Mass: 69937.547 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P39923

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Non-polymers , 1 types, 28 molecules

#9: Chemical...
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 28 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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: Seh1-associated complex (SEAC) / Type: COMPLEX / Entity ID: #1-#8 / Source: NATURAL
Molecular weightValue: 2.09 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 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 defocus max: 1600 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategoryDetails
4cryoSPARC3.3.2CTF correctionPatch CTF correction
7UCSF Chimera1.16model fitting
8Coot0.9.8.1model fitting
10cryoSPARC3.3.2initial Euler assignmentAb initio job
11cryoSPARC3.3.2final Euler assignmentNon-uniform refinement
13cryoSPARC3.3.23D reconstruction
14PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 208379 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: AlphaFold predictions were used as starting models for Sea1, Sea2, Sea3, Sea4, Npr2 and Npr3. Rigid body fit was performed in Chimera, and manual building in Coot.
Atomic model building

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

IDPDB-IDAccession codeInitial refinement model-ID
13F3F

3f3f

3F3F1
23MZK

3mzk

3MZK2

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