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- EMDB-30535: Cryo-EM structure of Schizosaccharomyces pombe Atg9 -

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Basic information

Entry
Database: EMDB / ID: EMD-30535
TitleCryo-EM structure of Schizosaccharomyces pombe Atg9
Map data
Sample
  • Organelle or cellular component: Protein detergent complex (Atg9-LMNG ) treated with GraFix
    • Protein or peptide: Autophagy-related protein 9
  • Ligand: Lauryl Maltose Neopentyl Glycol
KeywordsAutophagy / membrane protein / UNKNOWN FUNCTION
Function / homology
Function and homology information


Macroautophagy / : / phospholipid scramblase activity / protein localization to phagophore assembly site / phagophore assembly site membrane / autophagy of mitochondrion / fungal-type vacuole membrane / phagophore assembly site / autophagosome / macroautophagy ...Macroautophagy / : / phospholipid scramblase activity / protein localization to phagophore assembly site / phagophore assembly site membrane / autophagy of mitochondrion / fungal-type vacuole membrane / phagophore assembly site / autophagosome / macroautophagy / cytoplasmic vesicle membrane / autophagy / protein transport / membrane => GO:0016020 / Golgi membrane / endoplasmic reticulum membrane
Similarity search - Function
Autophagy-related protein 9 / Autophagy protein ATG9
Similarity search - Domain/homology
Autophagy-related protein 9
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsMatoba K / Tsutsumi A
Funding support Japan, 9 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)18K06097 Japan
Japan Society for the Promotion of Science (JSPS)15K21608 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101001 (support number 0053) Japan
Japan Science and TechnologyJPMJCR13M7 Japan
Japan Science and TechnologyJPMJCR14M1 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101079 (support number 0739) Japan
Japan Society for the Promotion of Science (JSPS)25111004 Japan
Japan Society for the Promotion of Science (JSPS)18H03989 Japan
Japan Society for the Promotion of Science (JSPS)19H05707 Japan
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion.
Authors: Kazuaki Matoba / Tetsuya Kotani / Akihisa Tsutsumi / Takuma Tsuji / Takaharu Mori / Daisuke Noshiro / Yuji Sugita / Norimichi Nomura / So Iwata / Yoshinori Ohsumi / Toyoshi Fujimoto / ...Authors: Kazuaki Matoba / Tetsuya Kotani / Akihisa Tsutsumi / Takuma Tsuji / Takaharu Mori / Daisuke Noshiro / Yuji Sugita / Norimichi Nomura / So Iwata / Yoshinori Ohsumi / Toyoshi Fujimoto / Hitoshi Nakatogawa / Masahide Kikkawa / Nobuo N Noda /
Abstract: The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM) ...The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
History
DepositionSep 10, 2020-
Header (metadata) releaseOct 28, 2020-
Map releaseOct 28, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7d0i
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30535.png

Downloads & links

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Map

FileDownload / File: emd_30535.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.245 Å
Density
Contour LevelBy AUTHOR: 0.0161 / Movie #1: 0.025
Minimum - Maximum-0.15201147 - 0.28488213
Average (Standard dev.)0.00046703496 (±0.007139964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 273.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2451.2451.245
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z273.900273.900273.900
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.1520.2850.000

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Supplemental data

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Mask #1

Fileemd_30535_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_30535_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_30535_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Protein detergent complex (Atg9-LMNG ) treated with GraFix

EntireName: Protein detergent complex (Atg9-LMNG ) treated with GraFix
Components
  • Organelle or cellular component: Protein detergent complex (Atg9-LMNG ) treated with GraFix
    • Protein or peptide: Autophagy-related protein 9
  • Ligand: Lauryl Maltose Neopentyl Glycol

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Supramolecule #1: Protein detergent complex (Atg9-LMNG ) treated with GraFix

SupramoleculeName: Protein detergent complex (Atg9-LMNG ) treated with GraFix
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)

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Macromolecule #1: Autophagy-related protein 9

MacromoleculeName: Autophagy-related protein 9 / type: protein_or_peptide / ID: 1
Details: A point mutation, S687Y, in the unobserved regions, is an unexpected mutation during plasmid preparation. C-terminal residues is linker (Gly-Ser) and cleaved site (Leu-Glu-Val-Leu-Phe-Gln).
Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Molecular weightTheoretical: 82.742172 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MFYQPAQNKK QYDDLADIEA QNNVPNTQEV LEAWQESLDS DEDESSPLEE SNGFTISEHD DFVKSVPRKN NPTDLLYSGK LLDSDEPPS VHGNSSKVPS KHPSPSFPET TSLRNLQNGS KQKPALPNFN DPHFYNEDVT RSGHPNRSIY TQLPRNEFSN A RVLWNRLS ...String:
MFYQPAQNKK QYDDLADIEA QNNVPNTQEV LEAWQESLDS DEDESSPLEE SNGFTISEHD DFVKSVPRKN NPTDLLYSGK LLDSDEPPS VHGNSSKVPS KHPSPSFPET TSLRNLQNGS KQKPALPNFN DPHFYNEDVT RSGHPNRSIY TQLPRNEFSN A RVLWNRLS ARDRVLWRWA NVENLDSFLQ QVYTYYTGKG LSCIIVHRLF QILTVSFVIG FTTFITSCID WPAVTPHGSL AG VTKSQCI AQMSPITYLV LWLFLSFLLA LWIYYLTDIP RLWQMREFYI HALKIATADM PTVSWQRVLY RLLKLKNVNA LTA EDGRVV SLHNMKRLDA YAIANRIMRK DNYFIALINN GIINIELPLL HRRILTHTTE WNINWCIFNF VFDEQGQLRS AFRN PNSRK RLSEELRRRF IVAGFLNCLF APIVAIYLVI HNFFRYFNEY HKNPGALSTR RYTPLALWTF REYNELQHFF DERIN DSYA AASHYVSQFP DFNMIRLFKY ISFILGSFTA ILVIITVFDP ELMVTFEITK DRSVLFYLGL FGSLIAVSRS IIPDET LVF APEKALRRVI TFTHYMPGWW SDNMHSKAVQ QEFCSLYSYR IVNLLWEILG ILLTPVLLFF TFPSCSQDIV DFFREHT IN VEGVGYVCSY AVFQDNPPYE SVASLVQSRK ISPLIQNKPE LSRIYFYEQF NTEAPRRDLR GSLEVLFQ

UniProtKB: Autophagy-related protein 9

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Macromolecule #2: Lauryl Maltose Neopentyl Glycol

MacromoleculeName: Lauryl Maltose Neopentyl Glycol / type: ligand / ID: 2 / Number of copies: 12 / Formula: LMN
Molecular weightTheoretical: 1.005188 KDa
Chemical component information

ChemComp-AV0:
Lauryl Maltose Neopentyl Glycol

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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

Concentration0.5 mg/mL
BufferpH: 8.3
Component:
ConcentrationFormulaName
20.0 mMHEPESN-(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonic Acid
150.0 mMKClpotassium chloride
0.002 %LMNGLauryl Maltose Neopentyl Glycol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.01 kPa / Details: The grid was washed by acetone prior to use
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 58245
FSC plot (resolution estimation)

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