|Entry||Database: PDB / ID: 7jlo|
|Title||Cryo-EM structure of human ATG9A in amphipols|
|Components||Autophagy-related protein 9A|
|Keywords||MEMBRANE 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 species||Homo sapiens (human)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å|
|Authors||Maeda, S. / Otomo, T.|
|Funding support|| United States, 1items |
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
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A: Autophagy-related protein 9A
B: Autophagy-related protein 9A
C: Autophagy-related protein 9A
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
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Human ATG9A in amphipols A8-35 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT|
|Molecular weight||Experimental value: NO|
|Source (natural)||Organism: Homo sapiens (human)|
|Source (recombinant)||Organism: Spodoptera frugiperda (fall armyworm)|
|Buffer solution||pH: 7.5|
|Specimen||Conc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid type: UltrAuFoil R1.2/1.3|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 88 % / Chamber temperature: 277 K|
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Microscopy||Model: FEI TALOS ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: 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 holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Electron dose: 66 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: C3 (3 fold cyclic)|
|3D reconstruction||Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21710 / Symmetry type: POINT|
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