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- EMDB-43639: Cryo-EM structure of mammalian Thorase filament, 3-layer refinement -

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Basic information

Entry
Database: EMDB / ID: EMD-43639
TitleCryo-EM structure of mammalian Thorase filament, 3-layer refinement
Map dataSharpened map from focused refinement of one turn of Thorase filament.
Sample
  • Organelle or cellular component: Thorase filament
    • Complex: Thorase monomer
      • Protein or peptide: Outer mitochondrial transmembrane helix translocase
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
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 / mitochondrial outer membrane / postsynaptic 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.25 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_43639.png

Downloads & links

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Map

FileDownload / File: emd_43639.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from focused refinement of one turn of Thorase filament.
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)

Slices (1/2)

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-0.022879366 - 2.1199663
Average (Standard dev.)0.0017960608 (±0.031300843)
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_43639_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from focused refinement of one turn of Thorase filament.

Fileemd_43639_half_map_1.map
AnnotationHalf map 2 from focused refinement of one turn of Thorase filament.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 from focused refinement of one turn of Thorase filament.

Fileemd_43639_half_map_2.map
AnnotationHalf map 1 from focused refinement of one turn of Thorase filament.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: Thorase filament

SupramoleculeName: Thorase filament / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
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: #1
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 / Number of copies: 6 / 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)
Molecular weightTheoretical: 36.226664 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: IDPTRKQKVE AQKQAEKLMK QIGVKNVKLS EYEMSIAAHL VDPLNMHVTW SDIAGLDDVI TDLKDTVILP IKKKHLFENS RLLQPPKGV LLYGPPGCGK TLIAKATAKE AGCRFINLQP STLTDKWYGE SQKLAAAVFS LAIKLQPSII FIDEIDSFLR N RSSSDHEA ...String:
IDPTRKQKVE AQKQAEKLMK QIGVKNVKLS EYEMSIAAHL VDPLNMHVTW SDIAGLDDVI TDLKDTVILP IKKKHLFENS RLLQPPKGV LLYGPPGCGK TLIAKATAKE AGCRFINLQP STLTDKWYGE SQKLAAAVFS LAIKLQPSII FIDEIDSFLR N RSSSDHEA TAMMKAQFMS LWDGLDTDHS CQVIVMGATN RPQDLDSAIM RRMPTRFHIN QPALKQREAI LKLILKNENV DR HVDLLEV AQETDGFSGS DLKEMCRDAA LLCVREYVNS TSEESHDEDE IRPVQQQDLH RAIEKMKKSK DAAFQNVLTH VCL D

UniProtKB: Outer mitochondrial transmembrane helix translocase

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Macromolecule #2: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 2 / Number of copies: 6 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle 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)

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 / Pretreatment - Atmosphere: AIR
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

Particle selectionNumber selected: 2105264
Startup modelType of model: NONE
Details: cryoSPARC helical reconstruction was used to generate the initial model without using a reference model.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 75556
Initial angle assignmentType: OTHER / Software - Name: RELION
Details: Helical reconstruction was used to generate the initial alignments of the boxed helical segments.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Details: Final angular assignment was refined without using helical reconstruction.
Final 3D classificationNumber classes: 4 / Avg.num./class: 58653 / Software - Name: RELION
Details: Focused 3D classification within mask with local angular searches was used to select the final set of particles.
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
Output model

PDB-8vxt:
Cryo-EM structure of mammalian Thorase filament, 3-layer refinement

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