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- PDB-7jlp: cryo-EM structure of human ATG9A in nanodiscs -

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Basic information

Entry
Database: PDB / ID: 7jlp
Titlecryo-EM structure of human ATG9A in nanodiscs
ComponentsAutophagy-related protein 9A
KeywordsMEMBRANE PROTEIN / autophagy
Function / homology
Function and homology information


phospholipid scramblase activity / programmed necrotic cell death / nucleophagy / protein localization to phagophore assembly site / phagophore assembly site membrane / phagophore assembly site / bone morphogenesis / Macroautophagy / autophagosome assembly / autophagosome ...phospholipid scramblase activity / programmed necrotic cell death / nucleophagy / protein localization to phagophore assembly site / phagophore assembly site membrane / phagophore assembly site / bone morphogenesis / Macroautophagy / autophagosome assembly / autophagosome / mitochondrial membrane / trans-Golgi network / recycling endosome / recycling endosome membrane / late endosome / late endosome membrane / endosome / Golgi membrane / intracellular membrane-bounded organelle / endoplasmic reticulum membrane / Golgi apparatus / mitochondrion / membrane
Similarity search - Function
Autophagy-related protein 9 / Autophagy protein ATG9
Similarity search - Domain/homology
Chem-POV / Autophagy-related protein 9A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMaeda, S. / Otomo, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM092740 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Structure, lipid scrambling activity and role in autophagosome formation of ATG9A.
Authors: Shintaro Maeda / Hayashi Yamamoto / Lisa N Kinch / Christina M Garza / Satoru Takahashi / Chinatsu Otomo / Nick V Grishin / Stefano Forli / Noboru Mizushima / Takanori Otomo /
Abstract: De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we ...De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.
History
DepositionJul 30, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 28, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 11, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 16, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)234,59548
Polymers200,3923
Non-polymers34,20345
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Autophagy-related protein 9A / APG9-like 1 / mATG9


Mass: 66797.344 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATG9A, APG9L1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7Z3C6
#2: Chemical...
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 45 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
Has ligand of interestN

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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: Human ATG9A in MSP2N2 nanodiscs / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 88 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 73000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.15rc3_3435refinement
PHENIX1.15rc3_3435refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2Leginonimage acquisition
4GctfCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18276 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002613182
ELECTRON MICROSCOPYf_angle_d0.45717670
ELECTRON MICROSCOPYf_chiral_restr0.03771878
ELECTRON MICROSCOPYf_plane_restr0.0032136
ELECTRON MICROSCOPYf_dihedral_angle_d14.60797818

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