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- EMDB-45512: The WIPI3:TSC lysosomal docking complex (focused reconstruction; ... -

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Basic information

Entry
Database: EMDB / ID: EMD-45512
TitleThe WIPI3:TSC lysosomal docking complex (focused reconstruction; TSC1 N-terminus)
Map dataRaw final map
Sample
  • Complex: TSC:WIPI3 lysosomal recruitment complex
KeywordsTuberous sclerosis complex / tumour suppressor / GTPase-activating proteins (GAP) / TSC-mTORC pathway / ONCOPROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBayly-Jones C / Lupton CJ / D'Andrea L / Ellisdon AM
Funding support United States, Australia, 3 items
OrganizationGrant numberCountry
Department of Defense (DOD, United States)W81XWH-19-1-0182 United States
Australian Research Council (ARC)DE240100992 Australia
Australian Research Council (ARC)LE170100016 Australia
CitationJournal: Sci Adv / Year: 2024
Title: Structure of the human TSC:WIPI3 lysosomal recruitment complex.
Authors: Charles Bayly-Jones / Christopher J Lupton / Laura D'Andrea / Yong-Gang Chang / Gareth D Jones / Joel R Steele / Hari Venugopal / Ralf B Schittenhelm / Michelle L Halls / Andrew M Ellisdon /
Abstract: Tuberous sclerosis complex (TSC) is targeted to the lysosomal membrane, where it hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, inhibiting mechanistic target of ...Tuberous sclerosis complex (TSC) is targeted to the lysosomal membrane, where it hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, inhibiting mechanistic target of rapamycin complex 1 (mTORC1). Loss-of-function mutations in TSC cause TSC disease, marked by excessive tumor growth. Here, we overcome a high degree of continuous conformational heterogeneity to determine the 2.8-Å cryo-electron microscopy (cryo-EM) structure of the complete human TSC in complex with the lysosomal recruitment factor WD repeat domain phosphoinositide-interacting protein 3 (WIPI3). We discover a previously undetected amino-terminal TSC1 HEAT repeat dimer that clamps onto a single TSC wing and forms a phosphatidylinositol phosphate (PIP)-binding pocket, which specifically binds monophosphorylated PIPs. These structural advances provide a model by which WIPI3 and PIP-signaling networks coordinate to recruit TSC to the lysosomal membrane to inhibit mTORC1. The high-resolution TSC structure reveals previously unrecognized mutational hotspots and uncovers crucial insights into the mechanisms of TSC dysregulation in disease.
History
DepositionJun 27, 2024-
Header (metadata) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45512.png

Downloads & links

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Map

FileDownload / File: emd_45512.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRaw final map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.17 Å/pix.
x 256 pix.
= 299.878 Å		1.17 Å/pix.
x 256 pix.
= 299.878 Å		1.17 Å/pix.
x 256 pix.
= 299.878 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1714 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.5817434 - 1.4337724
Average (Standard dev.)0.0032784857 (±0.034482706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 299.8784 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45512_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map

Fileemd_45512_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map (A)

Fileemd_45512_half_map_1.map
AnnotationUnfiltered half map (A)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map (B)

Fileemd_45512_half_map_2.map
AnnotationUnfiltered half map (B)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TSC:WIPI3 lysosomal recruitment complex

EntireName: TSC:WIPI3 lysosomal recruitment complex
Components
  • Complex: TSC:WIPI3 lysosomal recruitment complex

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Supramolecule #1: TSC:WIPI3 lysosomal recruitment complex

SupramoleculeName: TSC:WIPI3 lysosomal recruitment complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 733 KDa

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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

Concentration1.4 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
250.0 mMNaClsodium chloride
2.0 mMDTTthreo-1,4-Dimercapto-2,3-butanediol

Details: 20 mM HEPES (pH 7.6), 250 mM NaCl, 2 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 12807 / Average exposure time: 3.71 sec. / Average electron dose: 46.54 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.01 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1176292 / Details: Template picking, blob picking, TOPAZ, crYOLO
Startup modelType of model: OTHER / Details: Ab initio
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 99927
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: cryoSPARC (ver. 4.0.1), RELION (ver. 4.0))
Final 3D classificationAvg.num./class: 200000 / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

9815

chain_id: A, source_name: AlphaFold, initial_model_type: in silico model

2574

chain_id: C, source_name: AlphaFold, initial_model_type: in silico model

5ejc

source_name: PDB, initial_model_type: experimental model

9c9i

chain_id: F, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 96.4 / Target criteria: Cross-correlation coefficient

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