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- PDB-7jlo: Cryo-EM structure of human ATG9A in amphipols -

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Basic information

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


late nucleophagy / protein localization to phagophore assembly site / phagophore assembly site / autophagy of mitochondrion / autophagosome assembly / autophagosome / late endosome membrane / recycling endosome / trans-Golgi network / protein transport ...late nucleophagy / protein localization to phagophore assembly site / phagophore assembly site / autophagy of mitochondrion / autophagosome assembly / autophagosome / late endosome membrane / recycling endosome / trans-Golgi network / protein transport / late endosome / endosome / intracellular membrane-bounded organelle / endoplasmic reticulum membrane / membrane / integral component of membrane
Autophagy-related protein 9
Autophagy-related protein 9A
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.
Validation Report
SummaryFull reportAbout validation report
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

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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


Theoretical massNumber of molelcules
Total (without water)200,3923
Polymers200,3923
Non-polymers00
Water0
1


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

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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 amphipols A8-35 / 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.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: UltrAuFoil R1.2/1.3
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 / 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

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: 21710 / Symmetry type: POINT

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