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- EMDB-26840: Focused refinement of the Raptor-TFEB-Rag-Ragulator complex with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26840
TitleFocused refinement of the Raptor-TFEB-Rag-Ragulator complex with Raptor mask
Map datalocal refinement of Raptor-TFEB-Rag-Ragulator with Raptor mask, the map is post-processed according to local resolution.
Sample
  • Complex: Raptor subunit of the Raptor-TFEB-Rag-Ragulator complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsCui Z / Hurley J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM111730 United States
CitationJournal: Nature / Year: 2023
Title: Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex.
Authors: Zhicheng Cui / Gennaro Napolitano / Mariana E G de Araujo / Alessandra Esposito / Jlenia Monfregola / Lukas A Huber / Andrea Ballabio / James H Hurley /
Abstract: The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its ...The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagC-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.
History
DepositionMay 3, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26840.png

Downloads & links

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Map

FileDownload / File: emd_26840.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal refinement of Raptor-TFEB-Rag-Ragulator with Raptor mask, the map is post-processed according to local resolution.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 320 pix.
= 336. Å		1.05 Å/pix.
x 320 pix.
= 336. Å		1.05 Å/pix.
x 320 pix.
= 336. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-4.1585536 - 6.785198
Average (Standard dev.)0.0019129934 (±0.0739864)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_26840_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26840_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 : Raptor subunit of the Raptor-TFEB-Rag-Ragulator complex

EntireName: Raptor subunit of the Raptor-TFEB-Rag-Ragulator complex
Components
  • Complex: Raptor subunit of the Raptor-TFEB-Rag-Ragulator complex

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Supramolecule #1: Raptor subunit of the Raptor-TFEB-Rag-Ragulator complex

SupramoleculeName: Raptor subunit of the Raptor-TFEB-Rag-Ragulator complex
type: complex / ID: 1 / Chimera: Yes / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: ab-initio reconstruction by cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 377569
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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