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- EMDB-43640: Cryo-EM structure of mammalian Thorase filament, global helical r... -

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Basic information

Entry
Database: EMDB / ID: EMD-43640
TitleCryo-EM structure of mammalian Thorase filament, global helical refinement
Map data
Sample
  • Organelle or cellular component: Thorase filament
    • Complex: Thorase monomer
      • Protein or peptide: Outer mitochondrial transmembrane helix translocase
Keywordsfilament / AAA-ATPase / mitochondria / transmembrane / PROTEIN TRANSPORT
Function / homology
Function and homology information


Class I peroxisomal membrane protein import / extraction of mislocalized protein from mitochondrial outer membrane / membrane protein dislocase activity / Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate / negative regulation of synaptic transmission, glutamatergic / peroxisomal membrane / regulation of postsynaptic neurotransmitter receptor internalization / positive regulation of receptor internalization / learning / memory ...Class I peroxisomal membrane protein import / extraction of mislocalized protein from mitochondrial outer membrane / membrane protein dislocase activity / Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate / negative regulation of synaptic transmission, glutamatergic / peroxisomal membrane / regulation of postsynaptic neurotransmitter receptor internalization / positive regulation of receptor internalization / learning / memory / postsynaptic membrane / mitochondrial outer membrane / postsynapse / glutamatergic synapse / ATP hydrolysis activity / mitochondrion / ATP binding
Similarity search - Function
: / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Outer mitochondrial transmembrane helix translocase
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodhelical reconstruction / cryo EM / Resolution: 5.5 Å
AuthorsDar MA / Louder RK / Umanah GK / Dawson TM / Dawson VL
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA000266 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA044123 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS099362 United States
CitationJournal: bioRxiv / Year: 2024
Title: Cryo-EM Structure of AAA + ATPase Thorase Reveals Novel Helical Filament Formation.
Authors: Mohamad Aasif Dar / Robert Louder / Marisol Cortes / Rong Chen / Qianqian Ma / Mayukh Chakrabarti / George K E Umanah / Ted M Dawson / Valina L Dawson /
Abstract: The AAA+ (ATPases associated with a variety of cellular activities) ATPase, Thorase, also known as ATAD1, plays multiple roles in synaptic plasticity, mitochondrial quality control and mTOR signaling ...The AAA+ (ATPases associated with a variety of cellular activities) ATPase, Thorase, also known as ATAD1, plays multiple roles in synaptic plasticity, mitochondrial quality control and mTOR signaling through disassembling protein complexes like AMPAR and mTORC1 in an ATP-dependent manner. The Oligomerization of Thorase is crucial for its disassembly and remodeling functions. We show that wild-type Thorase forms long helical filaments , dependent on ATP binding but not hydrolysis. We report the Cryogenic Electron Microscopy (cryo-EM) structure of the Thorase filament at a resolution of 4 Å, revealing the dimeric arrangement of the basic repeating unit that is formed through a distinct interface compared to the hexameric MSP1/ATAD1E193Q assembly. Structure-guided mutagenesis confirms the role of critical amino acid residues required for filament formation, oligomerization and disassembly of mTORC1 protein complex. Together, our data reveals a novel filament structure of Thorase and provides critical information that elucidates the mechanism underlying Thorase filament formation and Thorase-mediated disassembly of the mTORC1 complex.
History
DepositionFeb 6, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43640.png

Downloads & links

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Map

FileDownload / File: emd_43640.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.0017643229 - 2.4437575
Average (Standard dev.)0.0050734603 (±0.05160979)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43640_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_43640_half_map_1.map
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Histogram

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Half map: #2

Fileemd_43640_half_map_2.map
Projections & Slices
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Sample components

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Entire : Thorase filament

EntireName: Thorase filament
Components
  • Organelle or cellular component: Thorase filament
    • Complex: Thorase monomer
      • Protein or peptide: Outer mitochondrial transmembrane helix translocase

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Supramolecule #1: Thorase filament

SupramoleculeName: Thorase filament / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 360 KDa

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Supramolecule #2: Thorase monomer

SupramoleculeName: Thorase monomer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Outer mitochondrial transmembrane helix translocase

MacromoleculeName: Outer mitochondrial transmembrane helix translocase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: IDPTRKQKVE AQKQAEKLMK QIGVKNVKLS EYEMSIAAHL VDPLNMHVTW SDIAGLDDVI TDLKDTVILP IKKKHLFENS RLLQPPKGVL LYGPPGCGKT LIAKATAKEA GCRFINLQPS TLTDKWYGES QKLAAAVFSL AIKLQPSIIF IDEIDSFLRN RSSSDHEATA ...String:
IDPTRKQKVE AQKQAEKLMK QIGVKNVKLS EYEMSIAAHL VDPLNMHVTW SDIAGLDDVI TDLKDTVILP IKKKHLFENS RLLQPPKGVL LYGPPGCGKT LIAKATAKEA GCRFINLQPS TLTDKWYGES QKLAAAVFSL AIKLQPSIIF IDEIDSFLRN RSSSDHEATA MMKAQFMSLW DGLDTDHSCQ VIVMGATNRP QDLDSAIMRR MPTRFHINQP ALKQREAILK LILKNENVDR HVDLLEVAQE TDGFSGSDLK EMCRDAALLC VREYVNSTSE ESHDEDEIRP VQQQDLHRAI EKMKKSKDAA FQNVLTHVCL D

UniProtKB: Outer mitochondrial transmembrane helix translocase

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
100.0 mMTris hydrochloride
200.0 mMsodium chlorideNaCl
5.0 mMmagnesium chlorideMgCl2
5.0 mMTris(2-carboxyethyl)phosphineC9H15O6P
5.0 mMAdenosine 5'-(3-thiotriphosphate)C10H12Li4N5O12P3S

Details: 100 mM Tris-Cl pH 7.5, 200 mM NaCl, 5.0 mM MgCl2, 5.0 mM TCEP, 5.0 mM ATP-gamma-S
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time of 3.0 s, blotting force of 5.
DetailsThorase filaments were prepared by mixing purified Thorase monomers with 5 mM ATP-gamma-S, followed by 60 minutes incubation on ice.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 4 / Number real images: 28295 / Average exposure time: 4.0 sec. / Average electron dose: 50.0 e/Å2
Details: Images were collected in counting mode, with 40 frames per 4 second movie.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 46296 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 81000
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

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 30.0 Å
Applied symmetry - Helical parameters - Δ&Phi: 60 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 234613
Segment selectionNumber selected: 2105264 / Software - Name: cryoSPARC
Software - details: filament tracer was used to pick overlapping filament segments
Startup modelType of model: NONE
Details: cryoSPARC helical reconstruction was used to generate the initial model without using a reference model.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Details: Final angular assignment was refined using helical symmetry.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1_v4


Chain - Residue range: 66-355 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: Regions with pLDDT below 30 were trimmed from the initial model.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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