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- EMDB-15374: Cryo-EM structure of the SEA complex (Sea2-Sea3 focused map) -

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Basic information

Entry
Database: EMDB / ID: EMD-15374
TitleCryo-EM structure of the SEA complex (Sea2-Sea3 focused map)
Map dataDeepEMhancer sharpened map
Sample
  • Complex: Focused reconstruction on SEAC core subunits Sea2-Seh1 & Sea3-Sec13, with C2-symmetry expanded particles
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsTafur L / Loewith R
Funding supportEuropean Union, 2 items
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 11, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15374.png

Downloads & links

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Map

FileDownload / File: emd_15374.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 640 pix.
= 464.64 Å		0.73 Å/pix.
x 640 pix.
= 464.64 Å		0.73 Å/pix.
x 640 pix.
= 464.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.726 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.0017442622 - 1.7258337
Average (Standard dev.)0.00037090754 (±0.011640489)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 464.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15374_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Non-resampled DeepEMhancer sharpened map

Fileemd_15374_additional_1.map
AnnotationNon-resampled DeepEMhancer sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15374_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15374_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Focused reconstruction on SEAC core subunits Sea2-Seh1 & Sea3-Sec...

EntireName: Focused reconstruction on SEAC core subunits Sea2-Seh1 & Sea3-Sec13, with C2-symmetry expanded particles
Components
  • Complex: Focused reconstruction on SEAC core subunits Sea2-Seh1 & Sea3-Sec13, with C2-symmetry expanded particles

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Supramolecule #1: Focused reconstruction on SEAC core subunits Sea2-Seh1 & Sea3-Sec...

SupramoleculeName: Focused reconstruction on SEAC core subunits Sea2-Seh1 & Sea3-Sec13, with C2-symmetry expanded particles
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#11
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 283 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.2) / Software - details: Patch CTF correction
Startup modelType of model: OTHER / Details: Ab initio map
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Software - details: Non-uniform refinement / Details: C2-symmetry expanded particles / Number images used: 416670
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.3.2) / Software - details: Ab initio job / Details: Ab initio map
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2) / Software - details: Non-uniform refinement / Details: Non-uniform refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3f3f

chain_id: A

3mzk

chain_id: A
DetailsAlphaFold 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.
RefinementSpace: REAL / Protocol: AB INITIO MODEL

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