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- EMDB-3721: The structure of the COPI coat leaf in complex with the ArfGAP2 u... -

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Basic information

Entry
Database: EMDB / ID: EMD-3721
TitleThe structure of the COPI coat leaf in complex with the ArfGAP2 uncoating factor
Map dataThe structure of the COPI coat leaf in complex with ArfGAP2 uncoating factor
Sample
  • Complex: The structure of the COPI coat leaf
    • Complex: COPI coat complex
      • Protein or peptide: Coatomer subunit alpha
      • Protein or peptide: Coatomer subunit beta
      • Protein or peptide: Coatomer subunit beta'
      • Protein or peptide: Coatomer subunit delta
      • Protein or peptide: Coatomer subunit gamma-1
      • Protein or peptide: Coatomer subunit zeta-1
    • Complex: ADP-ribosylation factor 1
      • Protein or peptide: ADP-ribosylation factor 1
    • Complex: ADP-ribosylation factor GTPase-activating protein 2
      • Protein or peptide: ADP-ribosylation factor GTPase-activating protein 2
KeywordsCOPI / coated vesicles / ArfGAP2 / Transport protein
Function / homology
Function and homology information


COPI coating of Golgi vesicle / cerebellar Purkinje cell layer maturation / protein localization to cell leading edge / Synthesis of PIPs at the plasma membrane / protein localization to axon / COPI-dependent Golgi-to-ER retrograde traffic / VxPx cargo-targeting to cilium / COPI-mediated anterograde transport / Synthesis of PIPs at the Golgi membrane / Intra-Golgi traffic ...COPI coating of Golgi vesicle / cerebellar Purkinje cell layer maturation / protein localization to cell leading edge / Synthesis of PIPs at the plasma membrane / protein localization to axon / COPI-dependent Golgi-to-ER retrograde traffic / VxPx cargo-targeting to cilium / COPI-mediated anterograde transport / Synthesis of PIPs at the Golgi membrane / Intra-Golgi traffic / trans-Golgi Network Vesicle Budding / protein localization to Golgi membrane / Golgi localization / COPI-coated vesicle / pancreatic juice secretion / regulation of Golgi organization / organelle membrane contact site / COPI vesicle coat / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / COPI-mediated anterograde transport / Golgi vesicle transport / positive regulation of mitochondrial fusion / COPI-dependent Golgi-to-ER retrograde traffic / organelle transport along microtubule / regulation of fatty acid metabolic process / establishment of Golgi localization / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane transport / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / pigmentation / Golgi-associated vesicle / positive regulation of mitochondrial fission / endoplasmic reticulum-Golgi intermediate compartment / protein secretion / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / Neutrophil degranulation / GTPase activator activity / adult locomotory behavior / small monomeric GTPase / establishment of localization in cell / macroautophagy / protein kinase C binding / intracellular protein transport / hormone activity / protein transport / growth cone / Golgi membrane / axon / GTPase activity / mRNA binding / neuronal cell body / GTP binding / structural molecule activity / Golgi apparatus / endoplasmic reticulum / extracellular space / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Coatomer beta subunit, C-terminal / Coatomer beta subunit (COPB1) / Coatomer beta subunit, appendage platform domain / Coatomer beta C-terminal region / Coatomer beta subunit appendage platform / Coatomer, gamma subunit, appendage, Ig-like subdomain / Coatomer gamma subunit / Coatomer subunit gamma, C-terminal / Coatomer, gamma subunit, appendage domain superfamily / Coatomer subunit zeta ...Coatomer beta subunit, C-terminal / Coatomer beta subunit (COPB1) / Coatomer beta subunit, appendage platform domain / Coatomer beta C-terminal region / Coatomer beta subunit appendage platform / Coatomer, gamma subunit, appendage, Ig-like subdomain / Coatomer gamma subunit / Coatomer subunit gamma, C-terminal / Coatomer, gamma subunit, appendage domain superfamily / Coatomer subunit zeta / Coatomer gamma subunit appendage platform subdomain / Coatomer subunit gamma-1 C-terminal appendage platform / Coatomer delta subunit / Coatomer, alpha subunit, C-terminal / Coatomer subunit alpha / : / Coatomer (COPI) alpha subunit C-terminus / Coatomer beta' subunit (COPB2) / Coatomer, WD associated region / : / Coatomer WD associated region / Arf GTPase activating protein / ArfGAP domain superfamily / Putative GTPase activating protein for Arf / ARF GTPase-activating proteins domain profile. / Putative GTP-ase activating proteins for the small GTPase, ARF / ARFGAP/RecO-like zinc finger / Cytochrome cd1-nitrite reductase-like, haem d1 domain superfamily / ADP-ribosylation factor 1-5 / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Adaptor complexes medium subunit family / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Small GTPase superfamily, ARF type / Clathrin adaptor, appendage, Ig-like subdomain superfamily / small GTPase Arf family profile. / Sar1p-like members of the Ras-family of small GTPases / Small GTPase superfamily, ARF/SAR type / ADP-ribosylation factor family / Longin-like domain superfamily / ARF-like small GTPases; ARF, ADP-ribosylation factor / TBP domain superfamily / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Coatomer subunit beta' / ADP-ribosylation factor 1 / Coatomer subunit zeta-1 / ADP-ribosylation factor GTPase-activating protein 2 / Coatomer subunit delta / Coatomer subunit alpha / Coatomer subunit beta / Coatomer subunit gamma-1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Saccharomyces cerevisiae (brewer's yeast) / Rattus norvegicus (Norway rat)
Methodsubtomogram averaging / cryo EM / Resolution: 10.1 Å
AuthorsDodonova SO / Aderhold P
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research FoundationSFB638 (A16 and Z4) Germany
German Research FoundationWI 654/12-1 Germany
CitationJournal: Elife / Year: 2017
Title: 9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.
Authors: Svetlana O Dodonova / Patrick Aderhold / Juergen Kopp / Iva Ganeva / Simone Röhling / Wim J H Hagen / Irmgard Sinning / Felix Wieland / John A G Briggs /
Abstract: COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that ...COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.
History
DepositionMay 15, 2017-
Header (metadata) releaseMay 31, 2017-
Map releaseJun 28, 2017-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5nzs
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3721.png

Downloads & links

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Map

FileDownload / File: emd_3721.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe structure of the COPI coat leaf in complex with ArfGAP2 uncoating factor
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.78 Å/pix.
x 128 pix.
= 227.84 Å		1.78 Å/pix.
x 128 pix.
= 227.84 Å		1.78 Å/pix.
x 128 pix.
= 227.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.78 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.19006988 - 0.22196983
Average (Standard dev.)0.0003264124 (±0.025159746)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 227.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.781.781.78
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z227.840227.840227.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1900.2220.000

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Supplemental data

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Sample components

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Entire : The structure of the COPI coat leaf

EntireName: The structure of the COPI coat leaf
Components
  • Complex: The structure of the COPI coat leaf
    • Complex: COPI coat complex
      • Protein or peptide: Coatomer subunit alpha
      • Protein or peptide: Coatomer subunit beta
      • Protein or peptide: Coatomer subunit beta'
      • Protein or peptide: Coatomer subunit delta
      • Protein or peptide: Coatomer subunit gamma-1
      • Protein or peptide: Coatomer subunit zeta-1
    • Complex: ADP-ribosylation factor 1
      • Protein or peptide: ADP-ribosylation factor 1
    • Complex: ADP-ribosylation factor GTPase-activating protein 2
      • Protein or peptide: ADP-ribosylation factor GTPase-activating protein 2

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Supramolecule #1: The structure of the COPI coat leaf

SupramoleculeName: The structure of the COPI coat leaf / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7 / Details: The asymmetric unit of the COPI coat
Molecular weightTheoretical: 550 KDa

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Supramolecule #2: COPI coat complex

SupramoleculeName: COPI coat complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4, #6-#7
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: ADP-ribosylation factor 1

SupramoleculeName: ADP-ribosylation factor 1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #4: ADP-ribosylation factor GTPase-activating protein 2

SupramoleculeName: ADP-ribosylation factor GTPase-activating protein 2 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #8
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Coatomer subunit alpha

MacromoleculeName: Coatomer subunit alpha / type: protein_or_peptide / ID: 1 / Details: WSHPQFEK is a C-terminal Strep-Tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 142.53275 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MLTKFETKSA RVKGLSFHPK RPWILTSLHN GVIQLWDYRM CTLIDKFDEH DGPVRGIDFH KQQPLFVSGG DDYKIKVWNY KLRRCLFTL LGHLDYIRTT FFHHEYPWIL SASDDQTIRV WNWQSRTCVC VLTGHNHYVM CAQFHPSEDL VVSASLDQTV R VWDISGLR ...String:
MLTKFETKSA RVKGLSFHPK RPWILTSLHN GVIQLWDYRM CTLIDKFDEH DGPVRGIDFH KQQPLFVSGG DDYKIKVWNY KLRRCLFTL LGHLDYIRTT FFHHEYPWIL SASDDQTIRV WNWQSRTCVC VLTGHNHYVM CAQFHPSEDL VVSASLDQTV R VWDISGLR KKNLSPGAVE SDVRGITGVD LFGTTDAVVK HVLEGHDRGV NWAAFHPTMP LIVSGADDRQ VKIWRMNESK AW EVDTCRG HYNNVSCAVF HPRQELILSN SEDKSIRVWD MSKRTGVQTF RRDHDRFWVL AAHPNLNLFA AGHDGGMIVF KLE RERPAY AVHGNMLHYV KDRFLRQLDF NSSKDVAVMQ LRSGSKFPVF NMSYNPAENA VLLCTRASNL ENSTYDLYTI PKDA DSQNP DAPEGKRSSG LTAVWVARNR FAVLDRMHSL LIKNLKNEIT KKIQVPNCDE IFYAGTGNLL LRDADSITLF DVQQK RTLA SVKISKVKYV IWSADMSHVA LLAKHAIVIC NRKLDALCNI HENIRVKSGA WDESGVFIYT TSNHIKYAVT TGDHGI IRT LDLPIYVTRV KGNNVYCLDR ECRPRVLTID PTEFKFKLAL INRKYDEVLH MVRNAKLVGQ SIIAYLQKKG YPEVALH FV KDEKTRFSLA LECGNIEIAL EAAKALDDKN CWEKLGEVAL LQGNHQIVEM CYQRTKNFDK LSFLYLITGN LEKLRKMM K IAEIRKDMSG HYQNALYLGD VSERVRILKN CGQKSLAYLS AATHGLDEEA ESLKETFDPE KETIPDIDPN AKLLQPPAP IMPLDTNWPL LTVSKGFFEG SIASKGKGGA LAADIDIDTV GTEGWGEDAE LQLDEDGFVE APEGLGEDVL GKGQEEGGGW DVEEDLELP PELDVPSGVS GSAEDGFFVP PTKGTSPTQI WCNNSQLPVD HILAGSFETA MRLLHDQVGV IQFGPYKQLF L QTYARGRT TYQALPCLPS MYSYPNRNWK DAGLKNGVPA VGLKLNDLIQ RLQLCYQLTT VGKFEEAVEK FRSILLSVPL LV VDNKQEI AEAQQLITIC REYIVGLCME IERKKLPKET LDQQKRICEM AAYFTHSNLQ PVHMILVLRT ALNLFFKLKN FKT AATFAR RLLELGPKPE VAQQTRKILS ACEKNPTDAC QLNYDMHNPF DICAASYRPI YRGKPVEKCP LSGACYSPEF KGQI CRVTT VTEIGKDVIG LRISPLQFRL EVLFQGPSAW SHPQFEKGGG SGGGSGGSAW SHPQFEK

UniProtKB: Coatomer subunit alpha

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Macromolecule #2: Coatomer subunit beta

MacromoleculeName: Coatomer subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 109.148109 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHENL YFQGHMTAAE NVCYTLINVP MDSEPPSEIS LKNDLEKGDV KSKTEALKKV IIMILNGEKL PGLLMTIIRF VLPLQDHTI KKLLLVFWEI VPKTTPDGRL LHEMILVCDA YRKDLQHPNE FIRGSTLRFL CKLKEAELLE PLMPAIRACL E HRHSYVRR ...String:
MHHHHHHENL YFQGHMTAAE NVCYTLINVP MDSEPPSEIS LKNDLEKGDV KSKTEALKKV IIMILNGEKL PGLLMTIIRF VLPLQDHTI KKLLLVFWEI VPKTTPDGRL LHEMILVCDA YRKDLQHPNE FIRGSTLRFL CKLKEAELLE PLMPAIRACL E HRHSYVRR NAVLAIYTIY RNFEHLIPDA PELIHDFLVN EKDASCKRNA FMMLIHADQD RALDYLSTCI DQVQTFGDIL QL VIVELIY KVCHANPSER ARFIRCIYNL LQSSSPAVKY EAAGTLVTLS SAPTAIKAAA QCYIDLIIKE SDNNVKLIVL DRL VELKEH PAHERVLQDL VMDILRVLST PDLEVRKKTL QLALDLVSSR NVEELVIVLK KEVIKTNNVS EHEDTDKYRQ LLVR TLHSC SVRFPDMAAN VIPVLMEFLS DSNEAAAADV LEFVREAIQR FDNLRMLIVE KMLEVFHAIK SVKIYRGALW ILGEY CSTK EDIQSVMTEV RRSLGEIPIV ESEIKKEAGE LKPEEEITVG PVQKLVTEMG TYATQSALSS SRPTKKEEDR PPLRGF LLD GDFFVAASLA TTLTKIALRY VALVQEKKKQ NSFVAEAMLL MATILHLGKS SLPKKPITDD DVDRISLCLK VLSECSP LM NDIFNKECRQ SLSQMLSAKL EEEKLSQKKE SEKRNVTVQP DDPISFMQLT AKNEMNCKED QFQLSLLAAM GNTQRKEA A DPLASKLNKV TQLTGFSDPV YAEAYVHVNQ YDIVLDVLVV NQTSDTLQNC TLELATLGDL KLVEKPSPLT LAPHDFANI KANVKVASTE NGIIFGNIVY DVSGAASDRN CVVLSDIHID IMDYIQPATC TDAEFRQMWA EFEWENKVTV NTNMTDLNDY LQHILKSTN MKCLTPEKAL SGYCGFMAAN LYARSIFGED ALANVSIEKP VHQGPDAAVT GHIRIRAKSQ GMALSLGDKI N LSQKKTSL

UniProtKB: Coatomer subunit beta

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Macromolecule #3: Coatomer subunit beta'

MacromoleculeName: Coatomer subunit beta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 102.566078 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MPLRLDIKRK LTARSDRVKS VDLHPTEPWM LASLYNGSVC VWNHETQTLV KTFEVCDLPV RAAKFVARKN WVVTGADDMQ IRVFNYNTL ERVHMFEAHS DYIRCIAVHP TQPFILTSSD DMLIKLWDWD KKWSCSQVFE GHTHYVMQIV INPKDNNQFA S ASLDRTIK ...String:
MPLRLDIKRK LTARSDRVKS VDLHPTEPWM LASLYNGSVC VWNHETQTLV KTFEVCDLPV RAAKFVARKN WVVTGADDMQ IRVFNYNTL ERVHMFEAHS DYIRCIAVHP TQPFILTSSD DMLIKLWDWD KKWSCSQVFE GHTHYVMQIV INPKDNNQFA S ASLDRTIK VWQLGSSSPN FTLEGHEKGV NCIDYYSGGD KPYLISGADD RLVKIWDYQN KTCVQTLEGH AQNVSCASFH PE LPIIITG SEDGTVRIWH SSTYRLESTL NYGMERVWCV ASLRGSNNVA LGYDEGSIIV KLGREEPAMS MDANGKIIWA KHS EVQQAN LKAMGDTEIK DGERLPLAVK DMGSCEIYPQ TIQHNPNGRF VVVCGDGEYI IYTAMALRNK SFGSAQEFAW AHDS SEYAI RESNSIVKIF KNFKEKKSFK PDFGAESIYG GFLLGVRSVN GLAFYDWENT ELIRRIEIQP KHIFWSDSGE LVCIA TEES FFILKYLSEK VLAAQETHEG VTEDGIEDAF EVLGEIQEIV KTGLWVGDCF IYTSSVNRLN YYVGGEIVTI AHLDRT MYL LGYIPKDNRL YLGDKELNIV SYSLLVSVLE YQTAVMRRDF SMADKVLPTI PKEQRTRVAH FLEKQGFKQQ ALTVSTD PE HRFELALQLG ELKIAYQLAV EAESEQKWKQ LAELAISKCQ FSLAQECLHH AQDYGGLLLL ATASGNASMV NKLAEGAE R DGKNNVAFMS YFLQGKLDAC LELLIRTGRL PEAAFLARTY LPSQVSRVVK LWRENLSKVN QKAAESLADP TEYENLFPG LKEAFVVEEW VKETHADLWP AKQYPLVTPN EERNVMEEAK GFQPSRPTAQ QEPDGKPASS PVIMASQTTH KEEKSLLELE VDLDNLELE DIDTTDINLD EDILDD

UniProtKB: Coatomer subunit beta'

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Macromolecule #4: Coatomer subunit delta

MacromoleculeName: Coatomer subunit delta / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 57.30425 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVLLAAAVCT KAGKAIVSRQ FVEMTRTRIE GLLAAFPKLM NTGKQHTFVE TESVRYVYQP MEKLYMVLIT TKNSNILEDL ETLRLFSRV IPEYCRALEE NEISEHCFDL IFAFDEIVAL GYRENVNLAQ IRTFTEMDSH EEKVFRAVRE TQEREAKAEM R RKAKELQQ ...String:
MVLLAAAVCT KAGKAIVSRQ FVEMTRTRIE GLLAAFPKLM NTGKQHTFVE TESVRYVYQP MEKLYMVLIT TKNSNILEDL ETLRLFSRV IPEYCRALEE NEISEHCFDL IFAFDEIVAL GYRENVNLAQ IRTFTEMDSH EEKVFRAVRE TQEREAKAEM R RKAKELQQ ARRDAERQGK KAPGFGGFGS SAVSGGSTAA MITETIIETD KPKVAPAPAR PSGPSKALKL GAKGKEVDNF VD KLKSEGE TIMSSNMGKR TSEATKVHAP PINMESVHMK IEEKITLTCG RDGGLQNMEL HGMIMLRISD DKFGRIRLHV ENE DKKGVQ LQTHPNVDKK LFTAESLIGL KNPEKSFPVN SDVGVLKWRL QTTEESFIPL TINCWPSESG NGCDVNIEYE LQED NLELN DVVITIPLPS GVGAPVIGEI DGEYRHDSRR NTLEWCLPVI DAKNKSGSLE FSIPGQPNDF FPVQVSFISK KNYCN IQVT KVTQVDGNSP VRFSTETTFL VDKYEIL

UniProtKB: Coatomer subunit delta

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Macromolecule #5: ADP-ribosylation factor 1

MacromoleculeName: ADP-ribosylation factor 1 / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 20.552438 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGLFASKLFS NLFGNKEMRI LMVGLDGAGK TTVLYKLKLG EVITTIPTIG FNVETVQYKN ISFTVWDVGG QDRIRSLWRH YYRNTEGVI FVVDSNDRSR IGEAREVMQR MLNEDELRNA AWLVFANKQD LPEAMSAAEI TEKLGLHSIR NRPWFIQATC A TSGEGLYE GLEWLSNSLK NST

UniProtKB: ADP-ribosylation factor 1

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Macromolecule #6: Coatomer subunit gamma-1

MacromoleculeName: Coatomer subunit gamma-1 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 97.622703 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MLKKFDKKDE ESGGGSNPLQ HLEKSAVLQE ARVFNETPIN PRKCAHILTK ILYLINQGEH LGTTEATEAF FAMTKLFQSN DPTLRRMCY LTIKEMSCIA EDVIIVTSSL TKDMTGKEDN YRGPAVRALC QITDSTMLQA VERYMKQAIV DKVPSVSSSA L VSSLHLLK ...String:
MLKKFDKKDE ESGGGSNPLQ HLEKSAVLQE ARVFNETPIN PRKCAHILTK ILYLINQGEH LGTTEATEAF FAMTKLFQSN DPTLRRMCY LTIKEMSCIA EDVIIVTSSL TKDMTGKEDN YRGPAVRALC QITDSTMLQA VERYMKQAIV DKVPSVSSSA L VSSLHLLK CSFDVVKRWV NEAQEAASSD NIMVQYHALG LLYHVRKNDR LAVSKMISKF TRHGLKSPFA YCMMIRVASK QL EEEDGSR DSPLFDFIES CLRNKHEMVV YEAASAIVNL PGCSAKELAP AVSVLQLFCS SPKAALRYAA VRTLNKVAMK HPS AVTACN LDLENLVTDS NRSIATLAIT TLLKTGSESS IDRLMKQISS FMSEISDEFK VVVVQAISAL CQKYPRKHAV LMNF LFTML REEGGFEYKR AIVDCIISII EENSESKETG LSHLCEFIED CEFTVLATRI LHLLGQEGPK TNNPSKYIRF IYNRV VLEH EEVRAGAVSA LAKFGAQNEE MLPSILVLLK RCVMDDDNEV RDRATFYLNV LEQKQKALNA GYILNGLTVS IPGLEK ALQ QYTLEPSEKP FDLKSVPLAT TPMAEQRPES TATAAVKQPE KVAATRQEIF QEQLAAVPEF QGLGPLFKSS PEPVALT ES ETEYVIRCTK HTFSDHLVFQ FDCTNTLNDQ TLENVTVQME PTEAYEVLSY VPARSLPYNQ PGTCYTLVAL PTEDPTAV A CTFSCVMKFT VKDCDPNTGE IDEEGYEDEY VLEDLEVTVA DHIQKVMKVN FEAAWDEVGD EFEKEETFTL STIKTLEEA VGNIVKFLGM HPCERSDKVP ENKNTHTLLL AGVFRGGHDI LVRSRLLLLD TVTMQVTARS SEELPVDIIL ASVG

UniProtKB: Coatomer subunit gamma-1

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Macromolecule #7: Coatomer subunit zeta-1

MacromoleculeName: Coatomer subunit zeta-1 / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 20.218168 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MEALILEPSL YTVKAILILD NDGDRLFAKY YDDTYPSVKE QKAFEKNIFN KTHRTDSEIA LLEGLTVVYK SSIDLYFYVI GSSYENELM LMAVLNCLFD SLSQMLRKNV EKRALLENME GLFLAVDEIV DGGVILESDP QQVVHRVALR GEDVPLTEQT V SQVLQSAK EQIKWSLLR

UniProtKB: Coatomer subunit zeta-1

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Macromolecule #8: ADP-ribosylation factor GTPase-activating protein 2

MacromoleculeName: ADP-ribosylation factor GTPase-activating protein 2 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 56.629246 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAAGPSKSEI QTLFKRLRAI PTNKACFDCG AKSPSWASIT YGVFLCIDCS GVHRSLGVHL SFIRSTELDS NWSWLQLRCM QVGGNANAT AFFRQHGCLA NDANTKYNSR AAQMYREKIR QLGSTALARH GTDLWIDNMN SAPSHSPEKK DSDFFTEHTQ A PAWDTAAT ...String:
MAAGPSKSEI QTLFKRLRAI PTNKACFDCG AKSPSWASIT YGVFLCIDCS GVHRSLGVHL SFIRSTELDS NWSWLQLRCM QVGGNANAT AFFRQHGCLA NDANTKYNSR AAQMYREKIR QLGSTALARH GTDLWIDNMN SAPSHSPEKK DSDFFTEHTQ A PAWDTAAT DPSGTQQPAL PSESSSLAQP EPGPNTDLLG TSPQASLELK SSIIGKKKPA AAKKGLGAKK GLGAQKVSNQ SF TEIERQA QVAEKLREQQ AADAKKQAEE SMVASMRLAY QELQIDRKKE EKKLQNLEGK KREQAERLGM GLVSRSSISH SVL SEMQMI EQETPLSAKS SRSQLDLFDD VGTFASGPPK YKDNPFSLGE TFGSRWDSDA AWGMDRVEEK EPEVTISSIR PISE RTTSR REVESRISGL ESSEARQKFA GAKAISSDMF FGREVDSEYE ARSRLQQLSG SSAISSSDLF GDVDGAHGGG TVSLG NVLP TADIAQFKQG VKSVAGKMAV LANGVMNSLQ DRYGSY

UniProtKB: ADP-ribosylation factor GTPase-activating protein 2

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state3D array

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationNameFormula
50.0 mMHEPES
50.0 mMKOAc
1.0 mMMgCl2

Details: Protein-A conjugated 10 nm gold was added to the reaction mix in 1:6 volume ratio before plunge-freezing
GridModel: C-flat / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: Protochips C-flat MultiHole, 20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 296 K / Instrument: HOMEMADE PLUNGER
Details: The sample was applied onto glow-discharged (30 sec, 20 mA) C-flat (Protochips Inc.) multihole grids. The grids were blotted from the back side for 11 seconds at room temperature in a ...Details: The sample was applied onto glow-discharged (30 sec, 20 mA) C-flat (Protochips Inc.) multihole grids. The grids were blotted from the back side for 11 seconds at room temperature in a chamber at 85% humidity and plunge-frozen into liquid ethane using a manual plunger..
DetailsCOPI-coated vesicles were produced in vitro by incubating coatomer, Arf1, GTPgS, ARNO and GUVs in a total volume of 40 ul for 30 minutes at 37C

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
DetailsTomographic tilt series were acquired with the dose-symmetric tilt-scheme (Hagen et al., J Struct Biol. 2017)
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-5 / Number grids imaged: 1 / Average electron dose: 2.0 e/Å2
Details: Each of the images in the tilt series was low-pass filtered according to the electron-dose acquired by the sample (Grant and Grigorieff, 2015).
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: TOM Toolbox, AV3)
Details: After classification and multi-reference alignment, the final COPI-ArfGAP2 class contained 12372 asymmetric units, comprising in total approximately 65 % of the initial dataset.
Number subtomograms used: 12372
ExtractionNumber tomograms: 27 / Number images used: 105663 / Reference model: EMDB-2985 low pass filtered to 55A / Software: (Name: Amira, TOM Toolbox)
Details: The dataset consisted of 27 tomograms containing 690 vesicles and near-complete buds. Subtomograms were extracted from the surface of the vesicles.
Final 3D classificationNumber classes: 2 / Avg.num./class: 12000 / Software: (Name: TOM Toolbox, AV3)
Details: Multireference alignment and classification on the complete leaf structure was performed in order to identify the subpopulation of leaves with bound ArfGAP2. To do this, the subtomograms ...Details: Multireference alignment and classification on the complete leaf structure was performed in order to identify the subpopulation of leaves with bound ArfGAP2. To do this, the subtomograms were aligned against the reference from the COPI-ArfGAP2-dataset and from the COPI-dataset and divided into two classes based on cross-correlation. The average structures were generated for both classes.
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-5nzs:
The structure of the COPI coat leaf in complex with the ArfGAP2 uncoating factor

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