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- EMDB-22375: Cryo-EM structure of human ATG9A in amphipols -

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Basic information

Entry
Database: EMDB / ID: EMD-22375
TitleCryo-EM structure of human ATG9A in amphipols
Map dataprimary map
Sample
  • Organelle or cellular component: Human ATG9A in amphipols A8-35
    • Protein or peptide: Autophagy-related protein 9A
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
Autophagy-related protein 9A
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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.
History
DepositionJul 30, 2020-
Header (metadata) releaseOct 28, 2020-
Map releaseOct 28, 2020-
UpdateDec 16, 2020-
Current statusDec 16, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jlo
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22375.png

Downloads & links

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Map

FileDownload / File: emd_22375.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map
Voxel sizeX=Y=Z: 1.134 Å
Density
Contour LevelBy AUTHOR: 0.033 / Movie #1: 0.045
Minimum - Maximum-0.13560958 - 0.2555045
Average (Standard dev.)0.00066953857 (±0.009212163)
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-0.1360.2560.001

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Supplemental data

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Additional map: raw map

Fileemd_22375_additional_1.map
Annotationraw map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_22375_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_22375_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human ATG9A in amphipols A8-35

EntireName: Human ATG9A in amphipols A8-35
Components
  • Organelle or cellular component: Human ATG9A in amphipols A8-35
    • Protein or peptide: Autophagy-related protein 9A

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Supramolecule #1: Human ATG9A in amphipols A8-35

SupramoleculeName: Human ATG9A in amphipols A8-35 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Autophagy-related protein 9A

MacromoleculeName: Autophagy-related protein 9A / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 66.797344 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAQFDTEYQR LEASYSDSPP GEEDLLVHVA EGSKSPWHHI ENLDLFFSRV YNLHQKNGFT CMLIGEIFEL MQFLFVVAFT TFLVSCVDY DILFANKMVN HSLHPTEPVK VTLPDAFLPA QVCSARIQEN GSLITILVIA GVFWIHRLIK FIYNICCYWE I HSFYLHAL ...String:
MAQFDTEYQR LEASYSDSPP GEEDLLVHVA EGSKSPWHHI ENLDLFFSRV YNLHQKNGFT CMLIGEIFEL MQFLFVVAFT TFLVSCVDY DILFANKMVN HSLHPTEPVK VTLPDAFLPA QVCSARIQEN GSLITILVIA GVFWIHRLIK FIYNICCYWE I HSFYLHAL RIPMSALPYC TWQEVQARIV QTQKEHQICI HKRELTELDI YHRILRFQNY MVALVNKSLL PLRFRLPGLG EA VFFTRGL KYNFELILFW GPGSLFLNEW SLKAEYKRGG QRLELAQRLS NRILWIGIAN FLLCPLILIW QILYAFFSYA EVL KREPGA LGARCWSLYG RCYLRHFNEL EHELQSRLNR GYKPASKYMN CFLSPLLTLL AKNGAFFAGS ILAVLIALTI YDED VLAVE HVLTTVTLLG VTVTVCRSFI PDQHMVFCPE QLLRVILAHI HYMPDHWQGN AHRSQTRDEF AQLFQYKAVF ILEEL LSPI VTPLILIFCL RPRALEIIDF FRNFTVEVVG VGDTCSFAQM DVRQHGHPQW LSAGQTEASV YQQAEDGKTE LSLMHF AIT NPGWQPPRES TAFLG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration12 mg/mL
BufferpH: 7.5
GridModel: UltrAuFoil R1.2/1.3 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 88 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 73000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 21710
FSC plot (resolution estimation)

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