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- PDB-7d0i: Cryo-EM structure of Schizosaccharomyces pombe Atg9 -

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Basic information

Entry
Database: PDB / ID: 7d0i
TitleCryo-EM structure of Schizosaccharomyces pombe Atg9
ComponentsAutophagy-related protein 9
KeywordsUNKNOWN FUNCTION / Autophagy / membrane protein
Function / homology
Function and homology information


Macroautophagy / phospholipid scramblase activity / protein localization to phagophore assembly site / phagophore assembly site membrane / piecemeal microautophagy of the nucleus / phagophore assembly site / fungal-type vacuole membrane / reticulophagy / autophagosome assembly / mitophagy ...Macroautophagy / phospholipid scramblase activity / protein localization to phagophore assembly site / phagophore assembly site membrane / piecemeal microautophagy of the nucleus / phagophore assembly site / fungal-type vacuole membrane / reticulophagy / autophagosome assembly / mitophagy / autophagosome / cytoplasmic vesicle membrane / macroautophagy / 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 (fission yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsMatoba, K. / Tsutsumi, A. / Kikkawa, M. / Noda, N.N.
Funding support Japan, 9items
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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 28, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 11, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
B: Autophagy-related protein 9
D: Autophagy-related protein 9
F: Autophagy-related protein 9
H: Autophagy-related protein 9
J: Autophagy-related protein 9
L: Autophagy-related protein 9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)508,51518
Polymers496,4536
Non-polymers12,06212
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "E"
d_2ens_1chain "C"
d_3ens_1chain "A"
d_4ens_1chain "G"
d_5ens_1chain "I"
d_6ens_1chain "K"
d_1ens_2chain "H"
d_2ens_2chain "D"
d_3ens_2chain "F"
d_4ens_2chain "B"
d_5ens_2chain "J"
d_6ens_2chain "L"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LMNLMNG
d_12ens_1LMNLMNH
d_21ens_1LMNLMND
d_22ens_1LMNLMNE
d_31ens_1LMNLMNA
d_32ens_1LMNLMNB
d_41ens_1LMNLMNJ
d_42ens_1LMNLMNK
d_51ens_1LMNLMNM
d_52ens_1LMNLMNN
d_61ens_1LMNLMNP
d_62ens_1LMNLMNQ
d_11ens_2TRPGLNL1 - 424
d_21ens_2TRPGLNF1 - 424
d_31ens_2TRPGLNI1 - 424
d_41ens_2TRPGLNC1 - 424
d_51ens_2TRPGLNO1 - 424
d_61ens_2TRPGLNR1 - 424

NCS ensembles :
ID
ens_1
ens_2

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Components

#1: Protein
Autophagy-related protein 9


Mass: 82742.172 Da / Num. of mol.: 6 / Mutation: S687Y
Source method: isolated from a genetically manipulated source
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).
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Gene: atg9, apg9, SPBC15D4.07c / Plasmid: pRS426-GAL1promoter / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): BJ926 diploid / References: UniProt: O74312
#2: Chemical
ChemComp-LMN / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside


Mass: 1005.188 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C47H88O22 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Protein detergent complex (Atg9-LMNG ) treated with GraFix
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ926 / Plasmid: pRS426
Buffer solutionpH: 8.3
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMN-(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonic AcidHEPES1
2150 mMpotassium chlorideKCl1
30.002 %Lauryl Maltose Neopentyl GlycolLMNG1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was washed by acetone prior to use / Grid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18_3855refinement
PHENIX1.18_3855refinement
EM softwareName: RELION / Version: 3.1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58245 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 42.22 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007222842
ELECTRON MICROSCOPYf_angle_d0.628931068
ELECTRON MICROSCOPYf_chiral_restr0.04613558
ELECTRON MICROSCOPYf_plane_restr0.00443678
ELECTRON MICROSCOPYf_dihedral_angle_d21.79967956
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2DELECTRON MICROSCOPYNCS constraints0.00143742594367
ens_1d_3BELECTRON MICROSCOPYNCS constraints0.00137530843055
ens_1d_4HELECTRON MICROSCOPYNCS constraints0.00148518667586
ens_1d_5JELECTRON MICROSCOPYNCS constraints0.00145128843814
ens_1d_6LELECTRON MICROSCOPYNCS constraints0.00136777621302
ens_2d_2DELECTRON MICROSCOPYNCS constraints0.00156860639104
ens_2d_3FELECTRON MICROSCOPYNCS constraints0.00146040647943
ens_2d_4BELECTRON MICROSCOPYNCS constraints0.00143459953762
ens_2d_5JELECTRON MICROSCOPYNCS constraints0.00151023360819
ens_2d_6LELECTRON MICROSCOPYNCS constraints0.0532031247236

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