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- PDB-8poe: Structure of tissue-specific lipid scramblase ATG9B homotrimer, r... -

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Basic information

Entry
Database: PDB / ID: 8poe
TitleStructure of tissue-specific lipid scramblase ATG9B homotrimer, refined with C3 symmetry applied
ComponentsAutophagy-related protein 9B
KeywordsLIPID TRANSPORT / membrane protein / lipid scramblase / autophagy / phagopore / lipid transporter / Atg9 / Atg9B
Function / homology
Function and homology information


phospholipid scramblase activity / programmed necrotic cell death / protein localization to phagophore assembly site / phagophore assembly site membrane / piecemeal microautophagy of the nucleus / bone morphogenesis / phagophore assembly site / reticulophagy / autophagy of mitochondrion / Macroautophagy ...phospholipid scramblase activity / programmed necrotic cell death / protein localization to phagophore assembly site / phagophore assembly site membrane / piecemeal microautophagy of the nucleus / bone morphogenesis / phagophore assembly site / reticulophagy / autophagy of mitochondrion / Macroautophagy / autophagosome assembly / autophagosome / trans-Golgi network / recycling endosome membrane / Golgi membrane / endoplasmic reticulum membrane
Similarity search - Function
Autophagy-related protein 9 / Autophagy protein ATG9
Similarity search - Domain/homology
Autophagy-related protein 9B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsChiduza, G.N. / Pye, V.E. / Tooze, S.A. / Cherepanov, P.
Funding support United Kingdom, European Union, 5items
OrganizationGrant numberCountry
Cancer Research UKCC2134, CC2058 United Kingdom
Medical Research Council (MRC, United Kingdom)CC2134, CC2058 United Kingdom
Wellcome TrustCC2134, CC2058 United Kingdom
European Research Council (ERC)788708European Union
The Francis Crick InstituteCC2134, CC2058 United Kingdom
CitationJournal: Autophagy / Year: 2024
Title: ATG9B is a tissue-specific homotrimeric lipid scramblase that can compensate for ATG9A.
Authors: George N Chiduza / Acely Garza-Garcia / Eugenia Almacellas / Stefano De Tito / Valerie E Pye / Alexander R van Vliet / Peter Cherepanov / Sharon A Tooze /
Abstract: Macroautophagy/autophagy is a fundamental aspect of eukaryotic biology, and the autophay-related protein ATG9A is part of the core machinery facilitating this process. In addition to ATG9A ...Macroautophagy/autophagy is a fundamental aspect of eukaryotic biology, and the autophay-related protein ATG9A is part of the core machinery facilitating this process. In addition to ATG9A vertebrates encode ATG9B, a poorly characterized paralog expressed in a subset of tissues. Herein, we characterize the structure of human ATG9B revealing the conserved homotrimeric quaternary structure and explore the conformational dynamics of the protein. Consistent with the experimental structure and computational chemistry, we establish that ATG9B is a functional lipid scramblase. We show that ATG9B can compensate for the absence of ATG9A in starvation-induced autophagy displaying similar subcellular trafficking and steady-state localization. Finally, we demonstrate that ATG9B can form a heteromeric complex with ATG2A. By establishing the molecular structure and function of ATG9B, our results inform the exploration of niche roles for autophagy machinery in more complex eukaryotes and reveal insights relevant across species. ATG: autophagy related; CHS: cholesteryl hemisuccinate; cryo-EM: single-particle cryogenic electron microscopy; CTF: contrast transfer function: CTH: C- terminal α helix; FSC: fourier shell correlation; HDIR: HORMA domain interacting region; LMNG: lauryl maltose neopentyl glycol; MD: molecular dynamics simulations; MSA: multiple sequence alignment; NBD-PE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl ammonium salt); POPC: palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RBG: repeating beta groove domain; RMSD: root mean square deviation; SEC: size-exclusion chromatography; TMH: transmembrane helix.
History
DepositionJul 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 29, 2023Group: Database references / Category: citation / Item: _citation.page_first / _citation.page_last
Revision 1.3Mar 20, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Autophagy-related protein 9B
B: Autophagy-related protein 9B
C: Autophagy-related protein 9B


Theoretical massNumber of molelcules
Total (without water)317,0013
Polymers317,0013
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Autophagy-related protein 9B / APG9-like 2 / Nitric oxide synthase 3-overlapping antisense gene protein / Protein sONE


Mass: 105667.008 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATG9B, APG9L2, NOS3AS / Production host: Homo sapiens (human) / References: UniProt: Q674R7

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Homotrimer of ATG9B / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.303 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8.5
Details: 20 mM Tris-HCl, pH 8.5; 200 mM NaCl; 1 mM TCEP; 10% glycerol supplemented with 0.001% w/v lauryl maltose neopentyl glycol and 0.0002% w/v cholesteryl hemisuccinate
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 50 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 46296 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.2 sec. / Electron dose: 48 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 37217
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1crYOLOparticle selection
2Gautomatchparticle selection
5GctfCTF correction
8Cootmodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6818472
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104050 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Cross-correlation coeffecient
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311235
ELECTRON MICROSCOPYf_angle_d0.61215306
ELECTRON MICROSCOPYf_dihedral_angle_d3.7031515
ELECTRON MICROSCOPYf_chiral_restr0.041761
ELECTRON MICROSCOPYf_plane_restr0.0041926

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