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- EMDB-17789: Structure of tissue-specific lipid scramblase ATG9B homotrimer, r... -

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Basic information

Entry
Database: EMDB / ID: EMD-17789
TitleStructure of tissue-specific lipid scramblase ATG9B homotrimer, refined with C3 symmetry applied
Map dataATG9B C3 map
Sample
  • Complex: Homotrimer of ATG9B
    • Protein or peptide: Autophagy-related protein 9B
Keywordsmembrane protein / lipid scramblase / autophagy / phagopore / lipid transporter / Atg9 / Atg9B / LIPID TRANSPORT
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 / trans-Golgi network / recycling endosome membrane / Golgi membrane / endoplasmic reticulum membrane
Similarity search - Function
Autophagy-related protein 9 / Autophagy protein ATG9
Similarity search - Domain/homology
Autophagy-related protein 9B
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsChiduza GN / Pye VE / Tooze SA / Cherepanov P
Funding support United Kingdom, European Union, 5 items
OrganizationGrant numberCountry
Cancer Research UKCC2134, CC2058 United Kingdom
Medical Research Council (MRC, United Kingdom)CC2134, CC2058 United Kingdom
Wellcome TrustCC2134, CC2058 United Kingdom
European Research Council (ERC)788708European Union
The Francis Crick InstituteCC2134, CC2058 United Kingdom
CitationJournal: Autophagy / Year: 2024
Title: ATG9B is a tissue-specific homotrimeric lipid scramblase that can compensate for ATG9A.
Authors: George N Chiduza / Acely Garza-Garcia / Eugenia Almacellas / Stefano De Tito / Valerie E Pye / Alexander R van Vliet / Peter Cherepanov / Sharon A Tooze /
Abstract: Macroautophagy/autophagy is a fundamental aspect of eukaryotic biology, and the autophay-related protein ATG9A is part of the core machinery facilitating this process. In addition to ATG9A ...Macroautophagy/autophagy is a fundamental aspect of eukaryotic biology, and the autophay-related protein ATG9A is part of the core machinery facilitating this process. In addition to ATG9A vertebrates encode ATG9B, a poorly characterized paralog expressed in a subset of tissues. Herein, we characterize the structure of human ATG9B revealing the conserved homotrimeric quaternary structure and explore the conformational dynamics of the protein. Consistent with the experimental structure and computational chemistry, we establish that ATG9B is a functional lipid scramblase. We show that ATG9B can compensate for the absence of ATG9A in starvation-induced autophagy displaying similar subcellular trafficking and steady-state localization. Finally, we demonstrate that ATG9B can form a heteromeric complex with ATG2A. By establishing the molecular structure and function of ATG9B, our results inform the exploration of niche roles for autophagy machinery in more complex eukaryotes and reveal insights relevant across species. ATG: autophagy related; CHS: cholesteryl hemisuccinate; cryo-EM: single-particle cryogenic electron microscopy; CTF: contrast transfer function: CTH: C- terminal α helix; FSC: fourier shell correlation; HDIR: HORMA domain interacting region; LMNG: lauryl maltose neopentyl glycol; MD: molecular dynamics simulations; MSA: multiple sequence alignment; NBD-PE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl ammonium salt); POPC: palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RBG: repeating beta groove domain; RMSD: root mean square deviation; SEC: size-exclusion chromatography; TMH: transmembrane helix.
History
DepositionJul 4, 2023-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17789.png

Downloads & links

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Map

FileDownload / File: emd_17789.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationATG9B C3 map
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.46
Minimum - Maximum-2.8552566 - 3.9496748
Average (Standard dev.)0.0038131839 (±0.07623518)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 302.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17789_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: ATG9B C3 DeepEMhancer map

Fileemd_17789_additional_1.map
AnnotationATG9B C3 DeepEMhancer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: ATG9B C3 half map 2

Fileemd_17789_half_map_1.map
AnnotationATG9B C3 half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: ATG9B C3 half map 1

Fileemd_17789_half_map_2.map
AnnotationATG9B C3 half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homotrimer of ATG9B

EntireName: Homotrimer of ATG9B
Components
  • Complex: Homotrimer of ATG9B
    • Protein or peptide: Autophagy-related protein 9B

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Supramolecule #1: Homotrimer of ATG9B

SupramoleculeName: Homotrimer of ATG9B / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 303 KDa

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Macromolecule #1: Autophagy-related protein 9B

MacromoleculeName: Autophagy-related protein 9B / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 105.667008 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DYKDDDDKDY KDDDDKDYKD DDDKHHHHHH ENLYFQGVSR MGWGGRRRRL GRWGDLGPGS VPLLPMPLPP PPPPSCRGPG GGRISIFSL SPAPHTRSSP SSFSPPTAGP PCSVLQGTGA SQSCHSALPI PATPPTQAQP AMTPASASPS WGSHSTPPLA P ATPTPSQQ ...String:
DYKDDDDKDY KDDDDKDYKD DDDKHHHHHH ENLYFQGVSR MGWGGRRRRL GRWGDLGPGS VPLLPMPLPP PPPPSCRGPG GGRISIFSL SPAPHTRSSP SSFSPPTAGP PCSVLQGTGA SQSCHSALPI PATPPTQAQP AMTPASASPS WGSHSTPPLA P ATPTPSQQ CPQDSPGLRV GPLIPEQDYE RLEDCDPEGS QDSPIHGEEQ QPLLHVPEGL RGSWHHIQNL DSFFTKIYSY HQ RNGFACI LLEDVFQLGQ FIFIVTFTTF LLRCVDYNVL FANQPSNHTR PGPFHSKVTL SDAILPSAQC AERIRSSPLL VLL LVLAAG FWLVQLLRSV CNLFSYWDIQ VFYREALHIP PEELSSVPWA EVQSRLLALQ RSGGLCVQPR PLTELDIHHR ILRY TNYQV ALANKGLLPA RCPLPWGGSA AFLSRGLALN VDLLLFRGPF SLFRGGWELP HAYKRSDQRG ALAARWGRTV LLLAA LNLA LSPLVLAWQV LHVFYSHVEL LRREPGALGA RGWSRLARLQ LRHFNELPHE LRARLARAYR PAAAFLRTAA PPAPLR TLL ARQLVFFAGA LFAALLVLTV YDEDVLAVEH VLTAMTALGV TATVARSFIP EEQCQGRAPQ LLLQTALAHM HYLPEEP GP GGRDRAYRQM AQLLQYRAVS LLEELLSPLL TPLFLLFWFR PRALEIIDFF HHFTVDVAGV GDICSFALMD VKRHGHPQ W LSAGQTEASL SQRAEDGKTE LSLMRFSLAH PLWRPPGHSS KFLGHLWGRV QQDAAAWGAT SARGPSTPGV LSNCTSPLP EAFLANLFVH PLLPPRDLSP TAPCPAAATA SLLASISRIA QDPSSVSPGG TGGQKLAQLP ELASAEMSLH VIYLHQLHQQ QQQQEPWGE AAASILSRPC SSPSQPPSPD EEKPSWSSDG SSPASSPRQQ WGTQKARNLF PGGFQVTTDT QKEPDRASCT D

UniProtKB: Autophagy-related protein 9B

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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

Concentration0.3 mg/mL
BufferpH: 8.5
Details: 20 mM Tris-HCl, pH 8.5; 200 mM NaCl; 1 mM TCEP; 10% glycerol supplemented with 0.001% w/v lauryl maltose neopentyl glycol and 0.0002% w/v cholesteryl hemisuccinate
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 50 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 46296 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 37217 / Average exposure time: 1.2 sec. / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6818472
Startup modelType of model: NONE
Details: Generated ab initio from selected 2D classes using cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 104050
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: OTHER / Target criteria: Cross-correlation coeffecient
Output model

PDB-8poe:
Structure of tissue-specific lipid scramblase ATG9B homotrimer, refined with C3 symmetry applied

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