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- EMDB-27183: Asymmetric unit of AP-1, Arf1, Nef lattice on MHC-I lipopeptide i... -

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Basic information

Entry
Database: EMDB / ID: EMD-27183
TitleAsymmetric unit of AP-1, Arf1, Nef lattice on MHC-I lipopeptide incorporated wide(r) membrane tubes
Map dataAsymmetric unit of AP-1, Arf1, Nef lattice on MHC-I lipopeptide incorporated wide(r) membrane tubes
Sample
  • Complex: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulated lipid bilayer
    • Complex: AP-1 heterotetramer
      • Protein or peptide: AP-1 complex subunit beta-1AP-1 transcription factor
      • Protein or peptide: AP-1 complex subunit gamma-1AP-1 transcription factor
      • Protein or peptide: AP-1 complex subunit mu-1AP-1 transcription factor
      • Protein or peptide: AP-1 complex subunit sigma-3AP-1 transcription factor
    • Protein or peptide: ADP-ribosylation factor 1ARF1
    • Protein or peptide: Protein Nef
    • Protein or peptide: HLA class I histocompatibility antigen, A alpha chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
Keywordsnef / AP / trafficking / PROTEIN TRANSPORT
Function / homology
Function and homology information


basolateral protein secretion / perturbation by virus of host immune response / negative regulation of CD4 production / mitotic cleavage furrow ingression / symbiont-mediated suppression of host T-cell mediated immune response / AP-1 adaptor complex / trans-Golgi Network Vesicle Budding / endosome to melanosome transport / positive regulation of natural killer cell degranulation / Lysosome Vesicle Biogenesis ...basolateral protein secretion / perturbation by virus of host immune response / negative regulation of CD4 production / mitotic cleavage furrow ingression / symbiont-mediated suppression of host T-cell mediated immune response / AP-1 adaptor complex / trans-Golgi Network Vesicle Budding / endosome to melanosome transport / positive regulation of natural killer cell degranulation / Lysosome Vesicle Biogenesis / platelet dense granule organization / protein trimerization / regulation of receptor internalization / melanosome assembly / regulation of Arp2/3 complex-mediated actin nucleation / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class I / Golgi to lysosome transport / Intra-Golgi traffic / Golgi to vacuole transport / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / Synthesis of PIPs at the Golgi membrane / Golgi Associated Vesicle Biogenesis / GTP-dependent protein binding / suppression by virus of host autophagy / clathrin adaptor activity / MHC class II antigen presentation / melanosome organization / thioesterase binding / CD4 receptor binding / Nef Mediated CD4 Down-regulation / dendritic spine organization / determination of left/right symmetry / long-term synaptic depression / COPI-dependent Golgi-to-ER retrograde traffic / clathrin-coated vesicle / Lysosome Vesicle Biogenesis / clathrin binding / Golgi Associated Vesicle Biogenesis / positive regulation of natural killer cell mediated cytotoxicity / host cell Golgi membrane / cell leading edge / Synthesis of PIPs at the plasma membrane / kinesin binding / T cell mediated cytotoxicity directed against tumor cell target / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-dependent / positive regulation of memory T cell activation / TAP complex binding / intracellular copper ion homeostasis / antigen processing and presentation of exogenous peptide antigen via MHC class I / Golgi medial cisterna / positive regulation of CD8-positive, alpha-beta T cell activation / CD8-positive, alpha-beta T cell activation / positive regulation of CD8-positive, alpha-beta T cell proliferation / protein targeting / CD8 receptor binding / MHC class I protein binding / endoplasmic reticulum exit site / beta-2-microglobulin binding / COPI-mediated anterograde transport / TAP binding / regulation of calcium-mediated signaling / clathrin-coated pit / protection from natural killer cell mediated cytotoxicity / vesicle-mediated transport / viral life cycle / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-independent / antigen processing and presentation of endogenous peptide antigen via MHC class Ib / MHC class II antigen presentation / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / detection of bacterium / Neutrophil degranulation / T cell receptor binding / sarcomere / small monomeric GTPase / trans-Golgi network membrane / Nef mediated downregulation of MHC class I complex cell surface expression / kidney development / virion component / lumenal side of endoplasmic reticulum membrane / Endosomal/Vacuolar pathway / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / intracellular protein transport / ER to Golgi transport vesicle membrane / cytoplasmic vesicle membrane / trans-Golgi network / peptide antigen assembly with MHC class I protein complex / MHC class I peptide loading complex / T cell mediated cytotoxicity / recycling endosome / antigen processing and presentation of endogenous peptide antigen via MHC class I / positive regulation of T cell cytokine production / cellular response to virus / small GTPase binding / MHC class I protein complex / SH3 domain binding / positive regulation of T cell mediated cytotoxicity / recycling endosome membrane / phagocytic vesicle membrane / peptide antigen binding / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell
Similarity search - Function
AP-1 complex subunit sigma / Adaptor protein complex AP-1, gamma subunit / Gamma-adaptin ear (GAE) domain / Gamma-adaptin ear (GAE) domain profile. / HIV-1 Nef protein, anchor domain superfamily / ADP-ribosylation factor 1-5 / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta ...AP-1 complex subunit sigma / Adaptor protein complex AP-1, gamma subunit / Gamma-adaptin ear (GAE) domain / Gamma-adaptin ear (GAE) domain profile. / HIV-1 Nef protein, anchor domain superfamily / ADP-ribosylation factor 1-5 / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta / Beta2-adaptin appendage, C-terminal sub-domain / Beta2-adaptin appendage, C-terminal sub-domain / HIV negative factor Nef / HIV-1 Nef protein, core domain superfamily / Negative factor, (F-Protein) or Nef / AP complex subunit beta / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / 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 adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Adaptin C-terminal domain / Adaptin C-terminal domain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / small GTPase Arf family profile. / Clathrin adaptor, appendage, Ig-like subdomain superfamily / 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 / Armadillo/beta-catenin-like repeats / Armadillo / MHC class I, alpha chain, C-terminal / MHC_I C-terminus / MHC class I alpha chain, alpha1 alpha2 domains / Class I Histocompatibility antigen, domains alpha 1 and 2 / Rab subfamily of small GTPases / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / MHC classes I/II-like antigen recognition protein / Armadillo-like helical / Small GTP-binding protein domain / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Armadillo-type fold / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
HLA class I histocompatibility antigen, A alpha chain / AP-1 complex subunit gamma-1 / AP-1 complex subunit mu-1 / ADP-ribosylation factor 1 / AP-1 complex subunit beta-1 / Protein Nef / AP-1 complex subunit sigma-3
Similarity search - Component
Biological speciesHomo sapiens (human) / Human immunodeficiency virus 1 / Mus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 9.2 Å
AuthorsHooy RM / Hurley JH
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)F32 AI152971 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI120691 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P50 AI150476 United States
CitationJournal: Sci Adv / Year: 2022
Title: Self-assembly and structure of a clathrin-independent AP-1:Arf1 tubular membrane coat.
Authors: Richard M Hooy / Yuichiro Iwamoto / Dan A Tudorica / Xuefeng Ren / James H Hurley /
Abstract: The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic whose mechanisms are unknown. HIV-1 Nef hijacks AP-1 to ...The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic whose mechanisms are unknown. HIV-1 Nef hijacks AP-1 to sequester major histocompatibility complex class I (MHC-I), evading immune detection. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes without clathrin and determined its structure. The coat assembles via Arf1 dimer interfaces. AP-1-positive tubules are enriched in cells upon clathrin knockdown. Nef localizes preferentially to AP-1 tubules in cells, explaining how Nef sequesters MHC-I. Coat contact residues are conserved across Arf isoforms and the Arf-dependent AP complexes AP-1, AP-3, and AP-4. Thus, AP complexes can self-assemble with Arf1 into tubular coats without clathrin or other scaffolding factors. The AP-1:Arf1 coat defines the structural basis of a broader class of tubulovesicular membrane coats as an intermediate in clathrin vesicle formation from internal membranes and as an MHC-I sequestration mechanism in HIV-1 infection.
History
DepositionJun 1, 2022-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27183.png

Downloads & links

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Map

FileDownload / File: emd_27183.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric unit of AP-1, Arf1, Nef lattice on MHC-I lipopeptide incorporated wide(r) membrane tubes
Voxel sizeX=Y=Z: 2.1 Å
Density
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-17.749417999999999 - 29.083199
Average (Standard dev.)-0.040340986 (±2.7180018)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 201.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27183_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 1

Fileemd_27183_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_27183_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulate...

EntireName: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulated lipid bilayer
Components
  • Complex: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulated lipid bilayer
    • Complex: AP-1 heterotetramer
      • Protein or peptide: AP-1 complex subunit beta-1AP-1 transcription factor
      • Protein or peptide: AP-1 complex subunit gamma-1AP-1 transcription factor
      • Protein or peptide: AP-1 complex subunit mu-1AP-1 transcription factor
      • Protein or peptide: AP-1 complex subunit sigma-3AP-1 transcription factor
    • Protein or peptide: ADP-ribosylation factor 1ARF1
    • Protein or peptide: Protein Nef
    • Protein or peptide: HLA class I histocompatibility antigen, A alpha chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulate...

SupramoleculeName: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulated lipid bilayer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Details: Asymmetric unit of the lattice on wide membrane tubes
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: AP-1 heterotetramer

SupramoleculeName: AP-1 heterotetramer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #4-#7
Details: All four subunits are co-expressed from the same plasmid. Assembly occurs in bacteria during expression.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: ADP-ribosylation factor 1

MacromoleculeName: ADP-ribosylation factor 1 / type: protein_or_peptide / ID: 1 / Details: N-terminal myristoylation / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.590547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GNIFANLFKG LFGKKEMRIL MVGLDAAGKT TILYKLKLGE IVTTIPTIGF NVETVEYKNI SFTVWDVGGQ DKIRPLWRHY FQNTQGLIF VVDSNDRERV NEAREELMRM LAEDELRDAV LLVFANKQDL PNAMNAAEIT DKLGLHSLRH RNWYIQATCA T SGDGLYEG LDWLSNQLRN QK

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Macromolecule #2: Protein Nef

MacromoleculeName: Protein Nef / type: protein_or_peptide / ID: 2 / Details: N-terminal myristoylation / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 24.154049 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GGKWSKSSVI GWPAVRERMR RAEPAADGVG AVSRDLEKHG AITSSNTAAN NAACAWLEAQ EEEEVGFPVT PQVPLRPMTY KAAVDLSHF LKEKGGLEGL IHSQRRQDIL DLWIYHTQGY FPDWQNYTPG PGVRYPLTFG WCYKLVPVEP DKVEEANKGE N TSLLHPVS ...String:
GGKWSKSSVI GWPAVRERMR RAEPAADGVG AVSRDLEKHG AITSSNTAAN NAACAWLEAQ EEEEVGFPVT PQVPLRPMTY KAAVDLSHF LKEKGGLEGL IHSQRRQDIL DLWIYHTQGY FPDWQNYTPG PGVRYPLTFG WCYKLVPVEP DKVEEANKGE N TSLLHPVS LHGMDDPERE VLEWRFDSRL AFHHVARELH PEYFKNCGHH HHHH

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Macromolecule #3: HLA class I histocompatibility antigen, A alpha chain

MacromoleculeName: HLA class I histocompatibility antigen, A alpha chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 4.139429 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
CRKSSDRKGG SYSQAAGSDS AQSSDVSLTA AKVHHHHHH

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Macromolecule #4: AP-1 complex subunit beta-1

MacromoleculeName: AP-1 complex subunit beta-1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 104.736461 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTDSKYFTTT KKGEIFELKA ELNSDKKEKK KEAVKKVIAS MTVGKDVSAL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN TFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQLVEDQ G FLDTLKDL ...String:
MTDSKYFTTT KKGEIFELKA ELNSDKKEKK KEAVKKVIAS MTVGKDVSAL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN TFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQLVEDQ G FLDTLKDL ISDSNPMVVA NAVAALSEIA ESHPSSNLLD LNPQSINKLL TALNECTEWG QIFILDCLAN YMPKDDREAQ SI CERVTPR LSHANSAVVL SAVKVLMKFM EMLSKDLDYY GTLLKKLAPP LVTLLSAEPE LQYVALRNIN LIVQKRPEIL KHE MKVFFV KYNDPIYVKL EKLDIMIRLA SQANIAQVLA ELREYATEVD VDFVRKAVRA IGRCAIKVEQ SAERCVSTLL DLIQ TKVNY VVQEAIVVIK DIFRKYPNKY ESVIATLCEN LDSLDEPEAR AAMIWIVGEY AERIDNADEL LESFLEGFHD KSTQV QLQL LTAIVKLFLK KPTETQELVQ QVLSLATQDS DNPDLRDRGY IYWRLLSTDP VAAKEVVLAE KPLISEETDL IEPTLL DEL ICYIGTLASV YHKPPSAFVE GGRGVVHKSL PPRTASSESA ESPETAPTGA PPGEQPDVIP AQGDLLGDLL NLDLGPP VS GPPLATSSVQ MGAVDLLGGG LDSLMGDEPE GIGGTNFVAP PTAAVPANLG APIGSGLSDL FDLTSGVGTL SGSYVAPK A VWLPAMKAKG LEISGTFTRQ VGSISMDLQL TNKALQVMTD FAIQFNRNSF GLAPAAPLQV HAPLSPNQTV EISLPLSTV GSVMKMEPLN NLQVAVKNNI DVFYFSTLYP LHILFVEDGK MDRQMFLATW KDIPNENEAQ FQIRDCPLNA EAASSKLQSS NIFTVAKRN VEGQDMLYQS LKLTNGIWVL AELRIQPGNP SCTDLELSLK CRAPEVSQHV YQAYETILKN

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Macromolecule #5: AP-1 complex subunit gamma-1

MacromoleculeName: AP-1 complex subunit gamma-1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 68.194094 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPAPIRLREL IRTIRTARTQ AEEREMIQKE CAAIRSSFRE EDNTYRCRNV AKLLYMHMLG YPAHFGQLEC LKLIASQKFT DKRIGYLGA MLLLDERQDV HLLMTNCIKN DLNHSTQFVQ GLALCTLGCM GSSEMCRDLA GEVEKLLKTS NSYLRKKAAL C AVHVIRKV ...String:
MPAPIRLREL IRTIRTARTQ AEEREMIQKE CAAIRSSFRE EDNTYRCRNV AKLLYMHMLG YPAHFGQLEC LKLIASQKFT DKRIGYLGA MLLLDERQDV HLLMTNCIKN DLNHSTQFVQ GLALCTLGCM GSSEMCRDLA GEVEKLLKTS NSYLRKKAAL C AVHVIRKV PELMEMFLPA TKNLLNEKNH GVLHTSVVLL TEMCERSPDM LAHFRKLVPQ LVRILKNLIM SGYSPEHDVS GI SDPFLQV RILRLLRILG RNDDDSSEAM NDILAQVATN TETSKNVGNA ILYETVLTIM DIKSESGLRV LAINILGRFL LNN DKNIRY VALTSLLKTV QTDHNAVQRH RSTIVDCLKD LDVSIKRRAM ELSFALVNGN NIRGMMKELL YFLDSCEPEF KADC ASGIF LAAEKYAPSK RWHIDTIMRV LTTAGSYVRD DAVPNLIQLI TNSVEMHAYT VQRLYKAILG DYSQQPLVQV AAWCI GEYG DLLVSGQCEE EEPIQVTEDE VLDILESVLI SNMSTSVTRG YALTAIMKLS TRFTCTVNRI KKVVSIYGSS IDVELQ QRA VEYNALFKKY DHMRSALLER MPVMEKVTTN GPENLYFQ

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Macromolecule #6: AP-1 complex subunit mu-1

MacromoleculeName: AP-1 complex subunit mu-1 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 48.475535 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SASAVYVLDL KGKVLICRNY RGDVDMSEVE HFMPILMEKE EEGMLSPILA HGGVRFMWIK HNNLYLVATS KKNACVSLVF SFLYKVVQV FSEYFKELEE ESIRDNFVII YELLDELMDF GYPQTTDSKI LQEYITQEGH KLETGAPRPP ATVTNAVSWR S EGIKYRKN ...String:
SASAVYVLDL KGKVLICRNY RGDVDMSEVE HFMPILMEKE EEGMLSPILA HGGVRFMWIK HNNLYLVATS KKNACVSLVF SFLYKVVQV FSEYFKELEE ESIRDNFVII YELLDELMDF GYPQTTDSKI LQEYITQEGH KLETGAPRPP ATVTNAVSWR S EGIKYRKN EVFLDVIEAV NLLVSANGNV LRSEIVGSIK MRVFLSGMPE LRLGLNDKVL FDNTGRGKSK SVELEDVKFH QC VRLSRFE NDRTISFIPP DGEFELMSYR LNTHVKPLIW IESVIEKHSH SRIEYMVKAK SQFKRRSTAN NVEIHIPVPN DAD SPKFKT TVGSVKWVPE NSEIVWSVKS FPGGKEYLMR AHFGLPSVEA EDKEGKPPIS VKFEIPYFTT SGIQVRYLKI IEKS GYQAL PWVRYITQNG DYQLRTQ

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Macromolecule #7: AP-1 complex subunit sigma-3

MacromoleculeName: AP-1 complex subunit sigma-3 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.305273 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MIHFILLFSR QGKLRLQKWY ITLPDKERKK ITREIVQIIL SRGHRTSSFV DWKELKLVYK RYASLYFCCA IENQDNELLT LEIVHRYVE LLDKYFGNVC ELDIIFNFEK AYFILDEFII GGEIQETSKK IAVKAIEDSD MLQEVSTVSQ TMGER

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Macromolecule #8: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 3 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.2
Component:
ConcentrationNameFormula
20.0 mMHEPES
125.0 mMpotassium acetateKOAc
2.0 mMmagnesium chloride
1.0 mMDTT

Details: HEPES/KOAc concentrated stocks are diluted to their final concentrations then pH'd to 7.2 with KOH prior to use in experiments.
GridModel: EMS Lacey Carbon / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 50
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: 60 second wait, 3-5 second blot, 597 filter paper, 0.5 second drain. Sample was supplemented with 10nm BSA-gold fiducials. 3.5ul of the mixture was double-side blotted..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Slit width: 25 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Average exposure time: 3.0 sec. / Average electron dose: 3.0 e/Å2
Details: Tilt images were collected in movie-mode. Each movie/tilt consisted of 3-4 frames each
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 24 / Number images used: 25534 / Reference model: Reference-free / Software - Name: Dynamo (ver. 1.1.532)
Details: Tubes were annotated by tracing the center of the tube in Dynamo and recording the average apparent diameter. Initial subtomogram positions were picked using uniform radial and axial sampling.
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.1) / Number subtomograms used: 18183
DetailsThe images were gain-normalized
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6cm9


4en2

chain_id: B

4en2

chain_id: E
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8d4e:
Asymmetric unit of AP-1, Arf1, Nef lattice on MHC-I lipopeptide incorporated wide(r) membrane tubes

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  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

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Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

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