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

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Basic information

Entry
Database: PDB / ID: 8vxt
TitleCryo-EM structure of mammalian Thorase filament, 3-layer refinement
ComponentsOuter mitochondrial transmembrane helix translocase
KeywordsPROTEIN TRANSPORT / filament / AAA-ATPase / mitochondria / transmembrane
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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Outer mitochondrial transmembrane helix translocase
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsDar, M.A. / Louder, R.K. / Umanah, G.K. / Dawson, T.M. / Dawson, V.L.
Funding support United States, 3items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Outer mitochondrial transmembrane helix translocase
B: Outer mitochondrial transmembrane helix translocase
C: Outer mitochondrial transmembrane helix translocase
D: Outer mitochondrial transmembrane helix translocase
E: Outer mitochondrial transmembrane helix translocase
F: Outer mitochondrial transmembrane helix translocase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)220,49912
Polymers217,3606
Non-polymers3,1396
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Helical is C2 symmetric, with 60-degree left-handed twist and 30 nm rise.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Outer mitochondrial transmembrane helix translocase / ATPase family AAA domain-containing protein 1 / Thorase


Mass: 36226.664 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Atad1 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9D5T0, Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate
#2: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Thorase filamentORGANELLE OR CELLULAR COMPONENT#10RECOMBINANT
2Thorase monomerCOMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
1124 kDa/nmNO
210.36 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Mus musculus (house mouse)10090
32Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)
32Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)
Buffer solutionpH: 7.5
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
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMTris hydrochloride1
2200 mMsodium chlorideNaCl1
35 mMmagnesium chlorideMgCl21
45 mMTris(2-carboxyethyl)phosphineC9H15O6P1
55 mMAdenosine 5'-(3-thiotriphosphate)1
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Thorase filaments were prepared by mixing purified Thorase monomers with 5 mM ATP-gamma-S, followed by 60 minutes incubation on ice.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blotting time of 3.0 s, blotting force of 5

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 46296 X / Nominal defocus max: 2000 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 4 / Num. of real images: 28295
Details: Images were collected in counting mode, with 40 frames per 4 second movie.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCparticle selectionfilament tracer was used to pick overlapping filament segments
2Latitudeimage acquisition
4RELIONCTF correction
7ISOLDEmodel fitting
9PHENIX1.20.1_4487:model refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2105264
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75556 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingAccession code: AF-Q8NBU5-F1_v4 / Chain residue range: 66-355
Details: Regions with pLDDT below 30 were trimmed from the initial model
Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314142
ELECTRON MICROSCOPYf_angle_d0.65919146
ELECTRON MICROSCOPYf_dihedral_angle_d9.4681896
ELECTRON MICROSCOPYf_chiral_restr0.0762190
ELECTRON MICROSCOPYf_plane_restr0.0052448

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