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- EMDB-27181: beta-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-27181
Titlebeta-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex within lattice on MHC-I lipopeptide incorporated narrow membrane tubes
Map databeta-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex within lattice on MHC-I lipopeptide incorporated narrow 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-1
      • Protein or peptide: AP-1 complex subunit gamma-1
      • Protein or peptide: AP-1 complex subunit mu-1
      • Protein or peptide: AP-1 complex subunit sigma-3
    • Protein or peptide: ADP-ribosylation factor 1
    • Protein or peptide: Protein Nef
    • Protein or peptide: HLA class I histocompatibility antigen, A alpha chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsnef / AP / trafficking / PROTEIN TRANSPORT
Function / homology
Function and homology information


basolateral protein secretion / endosome to melanosome transport / Lysosome Vesicle Biogenesis / AP-1 adaptor complex / mitotic cleavage furrow ingression / trans-Golgi Network Vesicle Budding / protein trimerization / platelet dense granule organization / negative regulation of glycoprotein biosynthetic process / Glycosphingolipid transport ...basolateral protein secretion / endosome to melanosome transport / Lysosome Vesicle Biogenesis / AP-1 adaptor complex / mitotic cleavage furrow ingression / trans-Golgi Network Vesicle Budding / protein trimerization / platelet dense granule organization / negative regulation of glycoprotein biosynthetic process / Glycosphingolipid transport / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class I / melanosome assembly / regulation of receptor internalization / Intra-Golgi traffic / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / regulation of Arp2/3 complex-mediated actin nucleation / Golgi Associated Vesicle Biogenesis / Synthesis of PIPs at the Golgi membrane / symbiont-mediated suppression of host autophagy / clathrin adaptor activity / symbiont-mediated suppression of host apoptosis / MHC class II antigen presentation / thioesterase binding / Nef Mediated CD4 Down-regulation / CD4 receptor binding / dendritic spine organization / long-term synaptic depression / determination of left/right symmetry / clathrin-coated vesicle / positive regulation of memory T cell activation / T cell mediated cytotoxicity directed against tumor cell target / TAP complex binding / COPI-dependent Golgi-to-ER retrograde traffic / 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 / clathrin binding / Lysosome Vesicle Biogenesis / Golgi Associated Vesicle Biogenesis / CD8 receptor binding / antigen processing and presentation of exogenous peptide antigen via MHC class I / beta-2-microglobulin binding / Synthesis of PIPs at the plasma membrane / cell leading edge / endoplasmic reticulum exit site / MHC class I protein binding / TAP binding / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-dependent / protection from natural killer cell mediated cytotoxicity / host cell Golgi membrane / 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 / intracellular copper ion homeostasis / detection of bacterium / protein targeting / T cell receptor binding / COPI-mediated anterograde transport / vesicle-mediated transport / viral life cycle / clathrin-coated pit / regulation of calcium-mediated signaling / Neutrophil degranulation / MHC class II antigen presentation / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / cytoplasmic vesicle membrane / Nef mediated downregulation of MHC class I complex cell surface expression / sarcomere / trans-Golgi network membrane / small monomeric GTPase / Endosomal/Vacuolar pathway / lumenal side of endoplasmic reticulum membrane / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / intracellular protein transport / kidney development / trans-Golgi network / ER to Golgi transport vesicle membrane / peptide antigen assembly with MHC class I protein complex / MHC class I peptide loading complex / T cell mediated cytotoxicity / positive regulation of T cell cytokine production / antigen processing and presentation of endogenous peptide antigen via MHC class I / MHC class I protein complex / cellular response to virus / peptide antigen binding / SH3 domain binding / positive regulation of T cell mediated cytotoxicity / virion component / positive regulation of type II interferon production / phagocytic vesicle membrane / recycling endosome membrane / Interferon gamma signaling / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / Interferon alpha/beta signaling / synaptic vesicle / antibacterial humoral response / T cell receptor signaling pathway / presynapse / E3 ubiquitin ligases ubiquitinate target proteins / heart development
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 / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta2-adaptin appendage, C-terminal sub-domain / HIV negative factor Nef / AP-1/2/4 complex subunit beta / HIV-1 Nef protein, core domain superfamily ...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 / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta2-adaptin appendage, C-terminal sub-domain / HIV negative factor Nef / AP-1/2/4 complex subunit beta / HIV-1 Nef protein, core domain superfamily / Negative factor, (F-Protein) or Nef / Adaptor protein complex, sigma subunit / : / Beta-adaptin appendage, C-terminal subdomain / Beta2-adaptin appendage, C-terminal sub-domain / ADP-ribosylation factor 1-5 / 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 / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Adaptin C-terminal domain / Adaptin C-terminal domain / AP complex, mu/sigma subunit / Adaptor complexes medium subunit family / Clathrin adaptor complex small chain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Small GTPase superfamily, ARF type / Small GTPase Arf domain 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 / 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 / : / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / Rab subfamily of small GTPases / 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: 9.3 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 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27181.png

Downloads & links

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Map

FileDownload / File: emd_27181.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationbeta-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex within lattice on MHC-I lipopeptide incorporated narrow membrane tubes
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
2.1 Å/pix.
x 128 pix.
= 268.8 Å		2.1 Å/pix.
x 128 pix.
= 268.8 Å		2.1 Å/pix.
x 128 pix.
= 268.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0
Minimum - Maximum-13.051023000000001 - 20.112162000000001
Average (Standard dev.)0.060735 (±2.0435562)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27181_msk_1.map
Projections & Slices
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Half map: Half Map 1

Fileemd_27181_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
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Half map: Half Map 2

Fileemd_27181_half_map_2.map
AnnotationHalf Map 2
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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-1
      • Protein or peptide: AP-1 complex subunit gamma-1
      • Protein or peptide: AP-1 complex subunit mu-1
      • Protein or peptide: AP-1 complex subunit sigma-3
    • Protein or peptide: ADP-ribosylation factor 1
    • Protein or peptide: Protein Nef
    • Protein or peptide: HLA class I histocompatibility antigen, A alpha chain
  • Ligand: GUANOSINE-5'-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: Dimeric contacts in lattice on narrow membrane tubes mediated by beta-Arf1 interactions
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: 4 / 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

UniProtKB: ADP-ribosylation factor 1

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Macromolecule #2: Protein Nef

MacromoleculeName: Protein Nef / type: protein_or_peptide / ID: 2 / Details: N-terminal myristoylation / Number of copies: 4 / 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

UniProtKB: Protein Nef

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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
Details: Conjugated to lipid maleimide via N-terminal cysteine
Number of copies: 2 / 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

UniProtKB: HLA class I histocompatibility antigen, A alpha chain

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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: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 104.605266 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: TDSKYFTTTK KGEIFELKAE LNSDKKEKKK EAVKKVIASM TVGKDVSALF PDVVNCMQTD NLELKKLVYL YLMNYAKSQP DMAIMAVNT FVKDCEDPNP LIRALAVRTM GCIRVDKITE YLCEPLRKCL KDEDPYVRKT AAVCVAKLHD INAQLVEDQG F LDTLKDLI ...String:
TDSKYFTTTK KGEIFELKAE LNSDKKEKKK EAVKKVIASM TVGKDVSALF PDVVNCMQTD NLELKKLVYL YLMNYAKSQP DMAIMAVNT FVKDCEDPNP LIRALAVRTM GCIRVDKITE YLCEPLRKCL KDEDPYVRKT AAVCVAKLHD INAQLVEDQG F LDTLKDLI SDSNPMVVAN AVAALSEIAE SHPSSNLLDL NPQSINKLLT ALNECTEWGQ IFILDCLANY MPKDDREAQS IC ERVTPRL SHANSAVVLS AVKVLMKFME MLSKDLDYYG TLLKKLAPPL VTLLSAEPEL QYVALRNINL IVQKRPEILK HEM KVFFVK YNDPIYVKLE KLDIMIRLAS QANIAQVLAE LREYATEVDV DFVRKAVRAI GRCAIKVEQS AERCVSTLLD LIQT KVNYV VQEAIVVIKD IFRKYPNKYE SVIATLCENL DSLDEPEARA AMIWIVGEYA ERIDNADELL ESFLEGFHDK STQVQ LQLL TAIVKLFLKK PTETQELVQQ VLSLATQDSD NPDLRDRGYI YWRLLSTDPV AAKEVVLAEK PLISEETDLI EPTLLD ELI CYIGTLASVY HKPPSAFVEG GRGVVHKSLP PRTASSESAE SPETAPTGAP PGEQPDVIPA QGDLLGDLLN LDLGPPV SG PPLATSSVQM GAVDLLGGGL DSLMGDEPEG IGGTNFVAPP TAAVPANLGA PIGSGLSDLF DLTSGVGTLS GSYVAPKA V WLPAMKAKGL EISGTFTRQV GSISMDLQLT NKALQVMTDF AIQFNRNSFG LAPAAPLQVH APLSPNQTVE ISLPLSTVG SVMKMEPLNN LQVAVKNNID VFYFSTLYPL HILFVEDGKM DRQMFLATWK DIPNENEAQF QIRDCPLNAE AASSKLQSSN IFTVAKRNV EGQDMLYQSL KLTNGIWVLA ELRIQPGNPS CTDLELSLKC RAPEVSQHVY QAYETILKN

UniProtKB: AP-1 complex subunit beta-1

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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: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 68.062891 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: PAPIRLRELI RTIRTARTQA EEREMIQKEC AAIRSSFREE DNTYRCRNVA KLLYMHMLGY PAHFGQLECL KLIASQKFTD KRIGYLGAM LLLDERQDVH LLMTNCIKND LNHSTQFVQG LALCTLGCMG SSEMCRDLAG EVEKLLKTSN SYLRKKAALC A VHVIRKVP ...String:
PAPIRLRELI RTIRTARTQA EEREMIQKEC AAIRSSFREE DNTYRCRNVA KLLYMHMLGY PAHFGQLECL KLIASQKFTD KRIGYLGAM LLLDERQDVH LLMTNCIKND LNHSTQFVQG LALCTLGCMG SSEMCRDLAG EVEKLLKTSN SYLRKKAALC A VHVIRKVP ELMEMFLPAT KNLLNEKNHG VLHTSVVLLT EMCERSPDML AHFRKLVPQL VRILKNLIMS GYSPEHDVSG IS DPFLQVR ILRLLRILGR NDDDSSEAMN DILAQVATNT ETSKNVGNAI LYETVLTIMD IKSESGLRVL AINILGRFLL NND KNIRYV ALTSLLKTVQ TDHNAVQRHR STIVDCLKDL DVSIKRRAME LSFALVNGNN IRGMMKELLY FLDSCEPEFK ADCA SGIFL AAEKYAPSKR WHIDTIMRVL TTAGSYVRDD AVPNLIQLIT NSVEMHAYTV QRLYKAILGD YSQQPLVQVA AWCIG EYGD LLVSGQCEEE EPIQVTEDEV LDILESVLIS NMSTSVTRGY ALTAIMKLST RFTCTVNRIK KVVSIYGSSI DVELQQ RAV EYNALFKKYD HMRSALLERM PVMEKVTTNG PENLYFQ

UniProtKB: AP-1 complex subunit gamma-1

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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: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 48.60673 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSASAVYVLD LKGKVLICRN YRGDVDMSEV EHFMPILMEK EEEGMLSPIL AHGGVRFMWI KHNNLYLVAT SKKNACVSLV FSFLYKVVQ VFSEYFKELE EESIRDNFVI IYELLDELMD FGYPQTTDSK ILQEYITQEG HKLETGAPRP PATVTNAVSW R SEGIKYRK ...String:
MSASAVYVLD LKGKVLICRN YRGDVDMSEV EHFMPILMEK EEEGMLSPIL AHGGVRFMWI KHNNLYLVAT SKKNACVSLV FSFLYKVVQ VFSEYFKELE EESIRDNFVI IYELLDELMD FGYPQTTDSK ILQEYITQEG HKLETGAPRP PATVTNAVSW R SEGIKYRK NEVFLDVIEA VNLLVSANGN VLRSEIVGSI KMRVFLSGMP ELRLGLNDKV LFDNTGRGKS KSVELEDVKF HQ CVRLSRF ENDRTISFIP PDGEFELMSY RLNTHVKPLI WIESVIEKHS HSRIEYMVKA KSQFKRRSTA NNVEIHIPVP NDA DSPKFK TTVGSVKWVP ENSEIVWSVK SFPGGKEYLM RAHFGLPSVE AEDKEGKPPI SVKFEIPYFT TSGIQVRYLK IIEK SGYQA LPWVRYITQN GDYQLRTQ

UniProtKB: AP-1 complex subunit mu-1

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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: 2 / 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

UniProtKB: AP-1 complex subunit sigma-3

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Macromolecule #8: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 4 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

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

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 4 / 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
Specialist opticsEnergy filter - Slit width: 25 eV
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
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: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 42000
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

DetailsThe images were gain-normalized
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 9.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.1) / Number subtomograms used: 7004
ExtractionNumber tomograms: 31 / Number images used: 61864 / 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
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6cm9

source_name: PDB, initial_model_type: experimental model

4en2

chain_id: B, source_name: PDB, initial_model_type: experimental model

4en2

chain_id: E, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8d4c:
beta-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex within lattice on MHC-I lipopeptide incorporated narrow membrane tubes

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