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- EMDB-44835: Cryo-EM map of the human autophagy tethering factor EPG5 -

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Basic information

Entry
Database: EMDB / ID: EMD-44835
TitleCryo-EM map of the human autophagy tethering factor EPG5
Map data
Sample
  • Complex: Ectopic P granules protein 5 homolog
    • Protein or peptide: Ectopic P granules protein 5 homolog
KeywordsTether / autophagy / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


nucleotide transport / maintenance of protein complex location / protein aggregate center assembly / interferon-mediated signaling pathway / inclusion body assembly / homeostasis of number of retina cells / cellular response to dsDNA / photoreceptor cell differentiation / response to type III interferon / host-mediated regulation of intestinal microbiota composition ...nucleotide transport / maintenance of protein complex location / protein aggregate center assembly / interferon-mediated signaling pathway / inclusion body assembly / homeostasis of number of retina cells / cellular response to dsDNA / photoreceptor cell differentiation / response to type III interferon / host-mediated regulation of intestinal microbiota composition / mucosal immune response / toll-like receptor 9 signaling pathway / endocytic recycling / endosome to lysosome transport / autophagosome maturation / response to unfolded protein / ubiquitin-dependent protein catabolic process / neuron apoptotic process / gene expression / defense response to virus / lysosome / inflammatory response / perinuclear region of cytoplasm / cytoplasm
Similarity search - Function
Ectopic P granules protein 5 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.85 Å
AuthorsCheung YWS / Nam SE / Yip CK
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Autophagy / Year: 2025
Title: Structure of the human autophagy factor EPG5 and the molecular basis of its conserved mode of interaction with Atg8-family proteins.
Authors: Yiu Wing Sunny Cheung / Sung-Eun Nam / Gage M J Fairlie / Karlton Scheu / Jennifer M Bui / Hannah R Shariati / Jörg Gsponer / Calvin K Yip /
Abstract: The multi-step macroautophagy/autophagy process ends with the cargo-laden autophagosome fusing with the lysosome to deliver the materials to be degraded. The metazoan-specific autophagy factor EPG5 ...The multi-step macroautophagy/autophagy process ends with the cargo-laden autophagosome fusing with the lysosome to deliver the materials to be degraded. The metazoan-specific autophagy factor EPG5 plays a crucial role in this step by enforcing fusion specificity and preventing mistargeting. How EPG5 exerts its critical function and how its deficiency leads to diverse phenotypes of the rare multi-system disorder Vici syndrome are not fully understood. Here, we report the first structure of human EPG5 (HsEPG5) determined by cryo-EM and AlphaFold2 modeling. Our structure revealed that HsEPG5 is constructed from helical bundles analogous to tethering factors in membrane trafficking pathways but contains a unique protruding thumb domain positioned adjacent to the atypical tandem LIR motifs involved in interaction with the GABARAP subfamily of Atg8-family proteins. Our NMR spectroscopic, molecular dynamics simulations and AlphaFold modeling studies showed that the HsEPG5 tandem LIR motifs only bind the canonical LIR docking site (LDS) on GABARAP without engaging in multivalent interaction. Our co-immunoprecipitation analysis further indicated that full-length HsEPG5-GABARAP interaction is mediated primarily by LIR1. Finally, our biochemical affinity isolation, X-ray crystallographic analysis, affinity measurement, and AlphaFold modeling demonstrated that this mode of binding is observed between EPG-5 and its Atg8-family proteins LGG-1 and LGG-2. Collectively our work generated novel insights into the structural properties of EPG5 and how it potentially engages with the autophagosome to confer fusion specificity.: ATG: autophagy related; CSP: chemical shift perturbation; eGFP: enhanced green fluoresent protein; EM: electron microscopy; EPG5: ectopic P-granules 5 autophagy tethering factor; GST: glutathione S-transferase; HP: hydrophobic pocket; HSQC: heteronuclear single-quantum correlation; ITC: isothermal titration calorimetry; LDS: LC3 docking site; LIR: LC3-interacting region; MD: molecular dynamics; NMR: nuclear magnetic resonance; TEV: tobacco etch virus.
History
DepositionMay 10, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44835.png

Downloads & links

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Map

FileDownload / File: emd_44835.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.4 Å/pix.
x 200 pix.
= 480. Å		2.4 Å/pix.
x 200 pix.
= 480. Å		2.4 Å/pix.
x 200 pix.
= 480. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.3300612 - 2.2750866
Average (Standard dev.)0.000016479371 (±0.047907818)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 480.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_44835_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44835_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44835_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : Ectopic P granules protein 5 homolog

EntireName: Ectopic P granules protein 5 homolog
Components
  • Complex: Ectopic P granules protein 5 homolog
    • Protein or peptide: Ectopic P granules protein 5 homolog

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Supramolecule #1: Ectopic P granules protein 5 homolog

SupramoleculeName: Ectopic P granules protein 5 homolog / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 298 KDa

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Macromolecule #1: Ectopic P granules protein 5 homolog

MacromoleculeName: Ectopic P granules protein 5 homolog / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMGSD YKDDDDKGSG I QRPTSTMA EAVKPQRRAK AK ASRTKTK EKKKYETPQR EES SEVSLP KTSREQEIPS LACE FKGDH LKVVTDSQLQ DDASG QNES EMFDVPLTSL TISNEE SLT CNTEPPKEGG EARPCVG DS ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMGSD YKDDDDKGSG I QRPTSTMA EAVKPQRRAK AK ASRTKTK EKKKYETPQR EES SEVSLP KTSREQEIPS LACE FKGDH LKVVTDSQLQ DDASG QNES EMFDVPLTSL TISNEE SLT CNTEPPKEGG EARPCVG DS AVTPKVHPGD NVGTKVET P KNFTEVEENM SVQGGLSES APQSNFSYTQ PAMENIQVRE TQNSKEDKQ GLVCSSEVPQ N VGLQSSCP AKHGFQTPRV KK LYPQLPA EIAGEAPALV AVK PLLRSE RLYPELPSQL ELVP FTKEQ LKILEPGSWL ENVES YLEE FDSMAHQDRH EFYELL LNY SRCRKQLLLA EAELLTL TS DCQNAKSRLW QFKEEQMS V QGICADQVKV FSYHRYQRV EMNENALVEL KKLFDAKSEH LHQTLALHS YTSVLSRLQV E SYIYALLS SSAVLRSSAI HQ QGRASKQ TESIPSDLCQ LKE CISVLF MFTRRVNEDT QFHD DILLW LQKLVSVLQR VGCPG DHLF LLNHILRCPA GVSKWA VPF IQIKVLHNPS GVFHFMQ SL ALLMSPVKNR AEFMCHMK P SERKPSSSGP GSGTWTLVD EGGEEDEDPE TSWILLNEDD LVTILAQFP FHELFQHLLG F KAKGDYLP ETTRPQEMMK IF AFANSLV ELLAVGLETF NRA RYRQFV KRIGYMIRMT LGYV SDHWA QYVSHNQGSG LAQQP YSME KLQVEFDELF LRAVLH VLK AKRLGIWLFM SEMPFGT LS VQMLWKLFYL MHQVESEN L QQLSSSLQPA QCKQQLQDP EHFTNFEKCL SSMNSSEEIC LLTTFAQMA QARRTNVDED F IKIIVLEI YEVSYVTLST RE TFSKVGR ELLGTITAVH PEI ISVLLD RVQETIDQVG MVSL YLFKE LPLYLWQPSA SEIAV IRDW LLNYNLTVVK NKLACV ILE GLNWGFAKQA TLHLDQA VH AEVALMVLEA YQKYLAQK P YAGILSESMK QVSYLASIV RYGETPETSF NQWAWNLILR LKLHKNDYG IQPNCPAVPF S VTVPDMTE SPTFHPLLKA VK AGMPIGC YLALSMTAVG HSI EKFCAE GIPLLGILVQ SRHL RTVVH VLDKILPLFY PCQYY LLKN EQFLSHLLLF LHLDSG VPQ GVTQQVTHKV AQHLTGA SH GDNVKLLNSM IQAHISVS T QPNEVGPVAV LEFWVQALI SQHLWYREQP ILFLMDHLCK AAFQLMQED CIQKLLYQQH K NALGYHCD RSLLSSLVSW IV AGNITPS FVEGLATPTQ VWF AWTVLN MESIFEEDSQ LRRV IEGEL VINSAFTPDQ ALKKA QTQL KLPIVPSLQR LLIYRW AHQ ALVTPSDHPL LPLIWQK FF LLYLHRPGPQ YGLPIDGC I GRRFFQSPAH INLLKEMKR RLTEVADFHH AASKALRVPA EGSEGLPES HSGTPGYLTS P ELHKELVR LFNVYILWLE DE NFQKGDT YIPSLPKHYD IHR LAKVMQ NQQDLWMEYL NMER IYHEF QETVGLWTQA KLESH STPC SLSVQLDFTD PLLAKE RVL SNLRKHEAPQ PPLALHP TK PPVPVISSAV LLSQKDAT Q LVCTDLNLLQ QQARTAALR ESQQVALDGE LLDTMPKQYV NREEQTTLH LECRGSSGKK C QGAAVVTV QFEGMHKNEA IS QQLHVLR KEVKQLQAEA AKP PSLNIV EAAVHAENLI TALV NAYKL QPTPGIQKVG ISLFF TIVD YVSDETQRHP PTRQFF TSC IEILGQVFIS GIKSECR KV LETILKNSRL CSLLSPFF T PNAAPAEFIQ LYEQVVKFL SEDNSDMIFM LLTKFDLKQW LSATKPPLS DRTRLLESIH L ALTAWGLE PDEDILMPFN LF CKHWTYL LLYQFPDQYS DIL RLLMQS SAEQLLSPEC WKAT LRALG CCAPSCQQGA ASTEG AVLP SSSDALLSDK QVMETI QWL SDFFYKLRLS KMDFKSF GL FSKWSPYMAD VKTFLGYL V KRLIDLEMTC LAQDPTASR KTVLKSLHSV IIQLFKPWIL VLEDNESSQ QRHYPWLESD T VVASSIVQ LFTDCIDSLH ES FKDKLLP GDAGALWLHL MHY CEACTA PKMPEFILYA FHST YRKLP WKDLHPDQML MEAFF KVER GSPKSCFLFL GSVLCE VNW VSVLSDAWNS SPHPETR SM IVCLLFMMIL LAKEVQLV D QTDSPLLSLL GQTSSLSWH LVDIVSYQSV LSYFSSHYPP SIILAKESY AELIMKLLKV S AGLSIPTD SQKHLDAVPK CQ AFTHQMV QFLSTLEQNG KIT LAVLEQ EMSKLLDDII VFNP PDMDS QTRHMALSSL FMEVL MMMN NATIPTAEFL RGSIRT WIG QKMHGLVVLP LLTAACQ SL ASVRHMAETT EACITAYF K ESPLNQNSGW GPILVSLQV PELTMEEFLQ ECLTLGSYLT LYVYLLQCL NSEQTLRNEM K VLLILSKW LEQVYPSSVE EE AKLFLWW HQVLQLSLIQ TEQ NDSVLT ESVIRILLLV QSRQ NLVAE ERLSSGILGA IGFGR KSPL SNRFRVVARS MAAFLS VQV PMEDQIRLRP GSELHLT PK AQQALNALES MASSKQYV E YQDQILQATQ FIRHPGHCL QDGKSFLALL VNCLYPEVHY LDHIR

UniProtKB: Ectopic P granules protein 5 homolog

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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

BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
150.0 mMNaClSodium chloride
0.01 %C32H58N2O7SCHAPS
0.5 mMC9H15O6PTCEP
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 875590
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: cryoSPARC ab initio reconstruction
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 103967
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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