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- EMDB-15605: Low resolution 3D reconstruction of ATG2A from cryo-EM -

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Basic information

Entry
Database: EMDB / ID: EMD-15605
TitleLow resolution 3D reconstruction of ATG2A from cryo-EM
Map data
Sample
  • Organelle or cellular component: Purified ATG2A
    • Protein or peptide: Autophagy-related protein 2 homolog A
KeywordsLipid transfer protein / autophagy / LIPID TRANSPORT
Function / homology
Function and homology information


phagophore / lipid transfer activity / organelle membrane contact site / glycophagy / positive regulation of autophagosome assembly / phagophore assembly site membrane / autophagy of mitochondrion / piecemeal microautophagy of the nucleus / pexophagy / phosphatidylinositol-3-phosphate binding ...phagophore / lipid transfer activity / organelle membrane contact site / glycophagy / positive regulation of autophagosome assembly / phagophore assembly site membrane / autophagy of mitochondrion / piecemeal microautophagy of the nucleus / pexophagy / phosphatidylinositol-3-phosphate binding / phagophore assembly site / reticulophagy / autophagosome assembly / protein-membrane adaptor activity / lipid droplet / positive regulation of autophagy / endoplasmic reticulum membrane
Similarity search - Function
Autophagy-related protein 2 / Autophagy-related protein 2
Similarity search - Domain/homology
Autophagy-related protein 2 homolog A
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.7 Å
AuthorsCherepanov P / Chiduza GN / Pye VE / van Vliet AR / Tooze SA
Funding support United Kingdom, European Union, 3 items
OrganizationGrant numberCountry
The Francis Crick InstituteFC001187 United Kingdom
European Research Council (ERC)788708European Union
European Molecular Biology Organization (EMBO)325-2017European Union
CitationJournal: Mol Cell / Year: 2022
Title: ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.
Authors: Alexander R van Vliet / George N Chiduza / Sarah L Maslen / Valerie E Pye / Dhira Joshi / Stefano De Tito / Harold B J Jefferies / Evangelos Christodoulou / Chloë Roustan / Emma Punch / ...Authors: Alexander R van Vliet / George N Chiduza / Sarah L Maslen / Valerie E Pye / Dhira Joshi / Stefano De Tito / Harold B J Jefferies / Evangelos Christodoulou / Chloë Roustan / Emma Punch / Javier H Hervás / Nicola O'Reilly / J Mark Skehel / Peter Cherepanov / Sharon A Tooze /
Abstract: ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow ...ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.
History
DepositionAug 18, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15605.png

Downloads & links

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Map

FileDownload / File: emd_15605.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.52 Å/pix.
x 140 pix.
= 352.8 Å		2.52 Å/pix.
x 140 pix.
= 352.8 Å		2.52 Å/pix.
x 140 pix.
= 352.8 Å

Surface

Projections

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Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.52 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0846
Minimum - Maximum-0.10575166 - 0.1984464
Average (Standard dev.)0.00043891725 (±0.0084527675)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 352.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15605_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15605_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15605_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 : Purified ATG2A

EntireName: Purified ATG2A
Components
  • Organelle or cellular component: Purified ATG2A
    • Protein or peptide: Autophagy-related protein 2 homolog A

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Supramolecule #1: Purified ATG2A

SupramoleculeName: Purified ATG2A / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Autophagy-related protein 2 homolog A

MacromoleculeName: Autophagy-related protein 2 homolog A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDYKDDDDKD YKDDDDKDYK DDDDKGSGAG AGAGAILNSR VSGLRSRMSR WLWPWSNCVK ERVCRYLLHH YLGHFFQEHL SLDQLSLDLY KGSVALRDIH LEIWSVNEVL ESMESPLELV EGFVGSIEVA VPWAALLTDH CTVRVSGLQL TLQPRRGPAP GAADSQSWAS ...String:
MDYKDDDDKD YKDDDDKDYK DDDDKGSGAG AGAGAILNSR VSGLRSRMSR WLWPWSNCVK ERVCRYLLHH YLGHFFQEHL SLDQLSLDLY KGSVALRDIH LEIWSVNEVL ESMESPLELV EGFVGSIEVA VPWAALLTDH CTVRVSGLQL TLQPRRGPAP GAADSQSWAS CMTTSLQLAQ ECLRDGLPEP SEPPQPLEGL EMFAQTIETV LRRIKVTFLD TVVRVEHSPG DGERGVAVEV RVQRLEYCDE AVRDPSQAPP VDVHQPPAFL HKLLQLAGVR LHYEELPAQE EPPEPPLQIG SCSGYMELMV KLKQNEAFPG PKLEVAGQLG SLHLLLTPRQ LQQLQELLSA VSLTDHEGLA DKLNKSRPLG AEDLWLIEQD LNQQLQAGAV AEPLSPDPLT NPLLNLDNTD LFFSMAGLTS SVASALSELS LSDVDLASSV RSDMASRRLS AQAHPAGKMA PNPLLDTMRP DSLLKMTLGG VTLTLLQTSA PSSGPPDLAT HFFTEFDATK DGPFGSRDFH HLRPRFQRAC PCSHVRLTGT AVQLSWELRT GSRGRRTTSM EVHFGQLEVL ECLWPRGTSE PEYTEILTFP GTLGSQASAR PCAHLRHTQI LRRVPKSRPR RSVACHCHSE LALDLANFQA DVELGALDRL AALLRLATVP AEPPAGLLTE PLPAMEQQTV FRLSAPRATL RLRFPIADLR PEPDPWAGQA VRAEQLRLEL SEPQFRSELS SGPGPPVPTH LELTCSDLHG IYEDGGKPPV PCLRVSKALD PKSTGRKYFL PQVVVTVNPQ SSSTQWEVAP EKGEELELSV ESPCELREPE PSPFSSKRTM YETEEMVIPG DPEEMRTFQS RTLALSRCSL EVILPSVHIF LPSKEVYESI YNRINNDLLM WEPADLLPTP DPAAQPSGFP GPSGFWHDSF KMCKSAFKLA NCFDLTPDSD SDDEDAHFFS VGASGGPQAA APEAPSLHLQ STFSTLVTVL KGRITALCET KDEGGKRLEA VHGELVLDME HGTLFSVSQY CGQPGLGYFC LEAEKATLYH RAAVDDYPLP SHLDLPSFAP PAQLAPTIYP SEEGVTERGA SGRKGQGRGP HMLSTAVRIH LDPHKNVKEF LVTLRLHKAT LRHYMALPEQ SWHSQLLEFL DVLDDPVLGY LPPTVITILH THLFSCSVDY RPLYLPVRVL ITAETFTLSS NIIMDTSTFL LRFILDDSAL YLSDKCEVET LDLRRDYVCV LDVDLLELVI KTWKGSTEGK LSQPLFELRC SNNVVHVHSC ADSCALLVNL LQYVMSTGDL HPPPRPPSPT EIAGQKLSES PASLPSCPPV ETALINQRDL ADALLDTERS LRELAQPSGG HLPQASPISV YLFPGERSGA PPPSPPVGGP AGSLGSCSEE KEDEREEEGD GDTLDSDEFC ILDAPGLGIP PRDGEPVVTQ LHPGPIVVRD GYFSRPIGST DLLRAPAHFP VPSTRVVLRE VSLVWHLYGG RDFGPHPGHR ARTGLSGPRS SPSRCSGPNR PQNSWRTQGG SGRQHHVLME IQLSKVSFQH EVYPAEPATG PAAPSQELEE RPLSRQVFIV QELEVRDRLA SSQINKFLYL HTSERMPRRA HSNMLTIKAL HVAPTTNLGG PECCLRVSLM PLRLNVDQDA LFFLKDFFTS LVAGINPVVP GETSAEARPE TRAQPSSPLE GQAEGVETTG SQEAPGGGHS PSPPDQQPIY FREFRFTSEV PIWLDYHGKH VTMDQVGTFA GLLIGLAQLN CSELKLKRLC CRHGLLGVDK VLGYALNEWL QDIRKNQLPG LLGGVGPMHS VVQLFQGFRD LLWLPIEQYR KDGRLMRGLQ RGAASFGSST ASAALELSNR LVQAIQATAE TVYDILSPAA PVSRSLQDKR SARRLRRGQQ PADLREGVAK AYDTVREGIL DTAQTICDVA SRGHEQKGLT GAVGGVIRQL PPTVVKPLIL ATEATSSLLG GMRNQIVPDA HKDHALKWRS DSAQDNSAVD GTAGPGSTGS R

UniProtKB: Autophagy-related protein 2 homolog A

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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

Concentration0.56 mg/mL
BufferpH: 8 / Details: 50 mM HEPES, 200 mM NaCl, 1 mM TCEP
GridModel: C-flat-1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
DetailsGrids prepared straight after size exclusion elution.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 8.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 14.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 42235
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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