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- EMDB-23120: 3D reconstruction of an autophagy tethering factor -

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Basic information

Entry
Database: EMDB / ID: EMD-23120
Title3D reconstruction of an autophagy tethering factor
Map dataEPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome-lysosome/late endosome fusion specificity.
Sample
  • Complex: Autophagy tethering factor, EPG5
    • Protein or peptide: EPG5, ectopic P-granules autophagy protein 5
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsNam SE / Yip CK
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Commun Biol / Year: 2021
Title: Insights on autophagosome-lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5.
Authors: Sung-Eun Nam / Yiu Wing Sunny Cheung / Thanh Ngoc Nguyen / Michael Gong / Samuel Chan / Michael Lazarou / Calvin K Yip /
Abstract: Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the ...Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome-lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended "shepherd's staff" architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome-lysosome fusion. The hEPG5-GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5's overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.
History
DepositionDec 15, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMar 17, 2021-
Current statusMar 17, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.581
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.581
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23120.png

Downloads & links

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Map

FileDownload / File: emd_23120.map.gz / Format: CCP4 / Size: 8.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome-lysosome/late endosome fusion specificity.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.6 Å/pix.
x 130 pix.
= 598. Å		4.6 Å/pix.
x 130 pix.
= 598. Å		4.6 Å/pix.
x 130 pix.
= 598. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.581 / Movie #1: 0.581
Minimum - Maximum-3.209552 - 12.64651
Average (Standard dev.)-0.016506562 (±0.47039768)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions130130130
Spacing130130130
CellA=B=C: 598.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.64.64.6
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z598.000598.000598.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ416416416
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS130130130
D min/max/mean-3.21012.647-0.017

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Supplemental data

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Sample components

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Entire : Autophagy tethering factor, EPG5

EntireName: Autophagy tethering factor, EPG5
Components
  • Complex: Autophagy tethering factor, EPG5
    • Protein or peptide: EPG5, ectopic P-granules autophagy protein 5

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Supramolecule #1: Autophagy tethering factor, EPG5

SupramoleculeName: Autophagy tethering factor, EPG5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
Molecular weightTheoretical: 297.83 kDa/nm

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Macromolecule #1: EPG5, ectopic P-granules autophagy protein 5

MacromoleculeName: EPG5, ectopic P-granules autophagy protein 5 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: MSYYHHHHHH DYDIPTTEN L YFQGAMGS DY KDDDDKG SGIQRPTSTM AEAVKPQRRA KAKASRTKTK EKKKYETPQR EESSEVSLPK TSREQEIPSL ACEFKGDHLK VVTDSQLQDD ASGQNESEMF DVPLTSLTIS NEESLTCNTE PPKEGGEARP CVGDSAVTPK ...String:
MSYYHHHHHH DYDIPTTEN L YFQGAMGS DY KDDDDKG SGIQRPTSTM AEAVKPQRRA KAKASRTKTK EKKKYETPQR EESSEVSLPK TSREQEIPSL ACEFKGDHLK VVTDSQLQDD ASGQNESEMF DVPLTSLTIS NEESLTCNTE PPKEGGEARP CVGDSAVTPK VHPGDNVGTK VETPKNFTEV EENMSVQGGL SESAPQSNFS YTQPAMENIQ VRETQNSKED KQGLVCSSEV PQNVGLQSSC PAKHGFQTPR VKKLYPQLPA EIAGEAPALV AVKPLLRSER LYPELPSQLE LVPFTKEQLK ILEPGSWLEN VESYLEEFDS MAHQDRHEFY ELLLNYSRC RKQLLLAEAE LLTLTSDCQN AKSRLWQFKE EQMSVQGICA DQVKVFSYHR YQRVEMNENA LVELKKLFD AKSEHLHQTL ALHSYTSVLS RLQVESYIYA LLSSSAVLRS SAIHQQGRAS K QTESIPSD LCQLKECISV LFMFTRRVNE DTQFHDDILL WLQKLVSVLQ RVGCPGDHLF LL NHILRCP AGVSKWAVPF IQIKVLHNPS GVFHFMQSLA LLMSPVKNRA EFMCHMKPSE RKP SSSGPG SGTWTLVDEG GEEDEDPETS WILLNEDDLV TILAQFPFHE LFQHLLGFKA KGDY LPETT RPQEMMKIFA FANSLVELLA VGLETFNRAR YRQFVKRIGY MIRMTLGYVS DHWAQ YVSH NQGSGLAQQP YSMEKLQVEF DELFLRAVLH VLKAKRLGIW LFMSEMPFGT LSVQML WKL FYLMHQVESE NLQQLSSSLQ PAQCKQQLQD PEHFTNFEKC LSSMNSSEEI CLLTTFA QM AQARRTNVDE DFIKIIVLEI YEVSYVTLST RETFSKVGRE LLGTITAVHP EIISVLLD R VQETIDQVGM VSLYLFKELP LYLWQPSASE IAVIRDWLLN YNLTVVKNKL ACVILEGLN WGFAKQATLH LDQAVHAEVA LMVLEAYQKY LAQKPYAGIL SESMKQVSYL ASIVRYGETP ETSFNQWAW NLILRLKLHK NDYGIQPNCP AVPFSVTVPD MTESPTFHPL LKAVKAGMPI G CYLALSMT AVGHSIEKFC AEGIPLLGIL VQSRHLRTVV HVLDKILPLF YPCQYYLLKN EQ FLSHLLL FLHLDSGVPQ GVTQQVTHKV AQHLTGASHG DNVKLLNSMI QAHISVSTQP NEV GPVAVL EFWVQALISQ HLWYREQPIL FLMDHLCKAA FQLMQEDCIQ KLLYQQHKNA LGYH CDRSL LSSLVSWIVA GNITPSFVEG LATPTQVWFA WTVLNMESIF EEDSQLRRVI EGELV INSA FTPDQALKKA QTQLKLPIVP SLQRLLIYRW AHQALVTPSD HPLLPLIWQK FFLLYL HRP GPQYGLPIDG CIGRRFFQSP AHINLLKEMK RRLTEVADFH HAASKALRVP AEGSEGL PE SHSGTPGYLT SPELHKELVR LFNVYILWLE DENFQKGDTY IPSLPKHYDI HRLAKVMQ N QQDLWMEYLN MERIYHEFQE TVGLWTQAKL ESHSTPCSLS VQLDFTDPLL AKERVLSNL RKHEAPQPPL ALHPTKPPVP VISSAVLLSQ KDATQLVCTD LNLLQQQART AALRESQQVA LDGELLDTM PKQYVNREEQ TTLHLECRGS SGKKCQGAAV VTVQFEGMHK NEAISQQLHV L RKEVKQLQ AEAAKPPSLN IVEAAVHAEN LITALVNAYK LQPTPGIQKV GISLFFTIVD YV SDETQRH PPTRQFFTSC IEILGQVFIS GIKSECRKVL ETILKNSRLC SLLSPFFTPN AAP AEFIQL YEQVVKFLSE DNSDMIFMLL TKFDLKQWLS ATKPPLSDRT RLLESIHLAL TAWG LEPDE DILMPFNLFC KHWTYLLLYQ FPDQYSDILR LLMQSSAEQL LSPECWKATL RALGC CAPS CQQGAASTEG AVLPSSSDAL LSDKQVMETI QWLSDFFYKL RLSKMDFKSF GLFSKW SPY MADVKTFLGY LVKRLIDLEM TCLAQDPTAS RKTVLKSLHS VIIQLFKPWI LVLEDNE SS QQRHYPWLES DTVVASSIVQ LFTDCIDSLH ESFKDKLLPG DAGALWLHLM HYCEACTA P KMPEFILYAF HSTYRKLPWK DLHPDQMLME AFFKVERGSP KSCFLFLGSV LCEVNWVSV LSDAWNSSPH PETRSMIVCL LFMMILLAKE VQLVDQTDSP LLSLLGQTSS LSWHLVDIVS YQSVLSYFS SHYPPSIILA KESYAELIMK LLKVSAGLSI PTDSQKHLDA VPKCQAFTHQ M VQFLSTLE QNGKITLAVL EQEMSKLLDD IIVFNPPDMD SQTRHMALSS LFMEVLMMMN NA TIPTAEF LRGSIRTWIG QKMHGLVVLP LLTAACQSLA SVRHMAETTE ACITAYFKES PLN QNSGWG PILVSLQVPE LTMEEFLQEC LTLGSYLTLY VYLLQCLNSE QTLRNEMKVL LILS KWLEQ VYPSSVEEEA KLFLWWHQVL QLSLIQTEQN DSVLTESVIR ILLLVQSRQN LVAEE RLSS GILGAIGFGR KSPLSNRFRV VARSMAAFLS VQVPMEDQIR LRPGSELHLT PKAQQA LNA LESMASSKQY VEYQDQILQA TQFIRHPGHC LQDGKSFLAL LVNCLYPEVH YLDHIR

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
StainingType: NEGATIVE / Material: Uranyl Formate
Sugar embeddingMaterial: Uranyl Formate
GridMaterial: COPPER / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal magnification: 49000
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 2)
Startup modelType of model: OTHER
Details: Ab initio was used to build initial model from 2D classification
Final reconstructionResolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 10752
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)

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