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- EMDB-22377: cryo-EM structure of human ATG9A in LMNG micelles -

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Basic information

Entry
Database: EMDB / ID: EMD-22377
Titlecryo-EM structure of human ATG9A in LMNG micelles
Map data
SampleHuman ATG9A in LMNG micelles:
Autophagy-related protein 9A
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsMaeda S / Otomo T
Funding support United States, 1 items
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 ReportPDB-ID: 7jlq

SummaryFull reportAbout validation report
History
DepositionJul 30, 2020-
Header (metadata) releaseOct 28, 2020-
Map releaseOct 28, 2020-
UpdateNov 11, 2020-
Current statusNov 11, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jlq
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22377.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.13 Å/pix.
x 192 pix.
= 217.728 Å
1.13 Å/pix.
x 192 pix.
= 217.728 Å
1.13 Å/pix.
x 192 pix.
= 217.728 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.134 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.02
Minimum - Maximum-0.08476854 - 0.13782012
Average (Standard dev.)-0.00016343294 (±0.0042825737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 217.728 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1341.1341.134
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z217.728217.728217.728
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0850.138-0.000

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Supplemental data

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Additional map: unsharpened

Fileemd_22377_additional_1.map
Annotationunsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half 2

Fileemd_22377_half_map_1.map
Annotationhalf 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half 1

Fileemd_22377_half_map_2.map
Annotationhalf 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Human ATG9A in LMNG micelles

EntireName: Human ATG9A in LMNG micelles / Number of components: 2

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Component #1: cellular-component, Human ATG9A in LMNG micelles

Cellular-componentName: Human ATG9A in LMNG micelles / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #2: protein, Autophagy-related protein 9A

ProteinName: Autophagy-related protein 9A / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 66.797344 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 3 mg/mL / pH: 7.5
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 88 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 66 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 73000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 1800.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 40116
3D reconstructionSoftware: RELION / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 7JLP
Output model

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