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- PDB-6dff: Structure of the cargo bound AP-1:Arf1:tetherin-Nef monomer -

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Basic information

Entry
Database: PDB / ID: 6dff
TitleStructure of the cargo bound AP-1:Arf1:tetherin-Nef monomer
Components
  • (AP-1 complex subunit ...AP-1 transcription factor) x 4
  • ADP-ribosylation factor 1ARF1
  • Bone marrow stromal antigen 2, Nef protein chimera
KeywordsTRANSPORT PROTEIN / AP / HIV / Nef / trafficking / VIRAL PROTEIN / PROTEIN TRANSPORT
Function / homology
Function and homology information


negative regulation of intracellular transport of viral material / negative regulation of plasmacytoid dendritic cell cytokine production / synaptic vesicle budding / phospholipase D activator activity / regulation of phospholipid metabolic process / positive regulation of late endosome to lysosome transport / lysosomal membrane organization / Golgi to transport vesicle transport / mitotic cleavage furrow ingression / negative regulation of CD4 biosynthetic process ...negative regulation of intracellular transport of viral material / negative regulation of plasmacytoid dendritic cell cytokine production / synaptic vesicle budding / phospholipase D activator activity / regulation of phospholipid metabolic process / positive regulation of late endosome to lysosome transport / lysosomal membrane organization / Golgi to transport vesicle transport / mitotic cleavage furrow ingression / negative regulation of CD4 biosynthetic process / suppression by symbiont of host T-cell mediated immune response / positive regulation of leukocyte proliferation / AP-1 adaptor complex / regulation of Arp2/3 complex-mediated actin nucleation / clathrin adaptor complex / positive regulation of natural killer cell degranulation / positive regulation of ER to Golgi vesicle-mediated transport / endosome to melanosome transport / Golgi to lysosome transport / response to interferon-beta / very-low-density lipoprotein particle assembly / melanosome organization / regulation of receptor internalization / suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I / response to interferon-alpha / COPI-coated vesicle / postsynaptic actin cytoskeleton organization / suppression by virus of host antigen processing and presentation of peptide antigen via MHC class II / suppression by virus of host adaptive immune response / metalloendopeptidase inhibitor activity / membrane coat / suppression by virus of host autophagy / positive regulation of calcium ion-dependent exocytosis / GTP-dependent protein binding / positive regulation of natural killer cell mediated cytotoxicity / long-term synaptic depression / CD4 receptor binding / determination of left/right symmetry / dendritic spine organization / thioesterase binding / regulation of calcium-mediated signaling / peroxisomal membrane / host cell Golgi membrane / azurophil granule membrane / clathrin binding / positive regulation of dendritic spine development / clathrin-coated vesicle / kinesin binding / MHC class I protein binding / response to interferon-gamma / positive regulation of endocytosis / B cell activation / cell leading edge / positive regulation of sodium ion transmembrane transport / negative regulation of viral genome replication / phosphatidylinositol biosynthetic process / cellular copper ion homeostasis / protein targeting / trans-Golgi network membrane / clathrin-coated pit / Rab GTPase binding / cellular response to virus / antigen processing and presentation of exogenous peptide antigen via MHC class II / clathrin-coated vesicle membrane / regulation of actin cytoskeleton organization / negative regulation of cell migration / anchored component of membrane / sarcomere / positive regulation of protein secretion / actin filament organization / vesicle-mediated transport / multivesicular body / microtubule cytoskeleton organization / collagen binding / interleukin-12-mediated signaling pathway / response to virus / microtubule cytoskeleton / GDP binding / type I interferon signaling pathway / recycling endosome / intracellular protein transport / virion / cytoplasmic vesicle membrane / trans-Golgi network / regulation of defense response to virus by virus / viral life cycle / SH3 domain binding / negative regulation of cell growth / lysosomal membrane / defense response to virus / late endosome / apical plasma membrane / heart development / positive regulation of I-kappaB kinase/NF-kappaB signaling / ATPase binding / protein C-terminus binding / postsynaptic density / GTPase activity / neuron projection / Golgi membrane
Small GTPase superfamily, ARF/SAR type / Clathrin adaptor, mu subunit, conserved site / Bone marrow stromal antigen 2 / HIV negative factor Nef / AP complex, mu/sigma subunit / TBP domain superfamily / Small GTP-binding protein domain / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Armadillo / P-loop containing nucleoside triphosphate hydrolase ...Small GTPase superfamily, ARF/SAR type / Clathrin adaptor, mu subunit, conserved site / Bone marrow stromal antigen 2 / HIV negative factor Nef / AP complex, mu/sigma subunit / TBP domain superfamily / Small GTP-binding protein domain / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Armadillo / P-loop containing nucleoside triphosphate hydrolase / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Adaptor protein complex AP-1, gamma subunit / Adaptor protein complex, sigma subunit / Clathrin adaptor complex, small chain / AP-1/2/4 complex subunit beta / Clathrin adaptor, mu subunit / AP complex subunit beta / HIV-1 Nef protein, anchor domain superfamily / Beta-adaptin appendage, C-terminal subdomain / Longin-like domain superfamily / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Gamma-adaptin ear (GAE) domain / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / Negative factor, (F-Protein) or Nef / Adaptin N terminal region / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / HIV-1 Nef protein, core domain superfamily / Armadillo-like helical / Armadillo-type fold / Beta-Lactamase - #60 / Leucine-rich Repeat Variant / Leucine-rich Repeat Variant / Beta-Lactamase / Alpha Horseshoe / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
AP-1 complex subunit beta-1 / Bone marrow stromal antigen 2 / AP-1 complex subunit gamma-1 / AP-1 complex subunit mu-1 / ADP-ribosylation factor 1 / Protein Nef / AP-1 complex subunit sigma-3
Biological speciesHomo sapiens (human)
Human immunodeficiency virus 1
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsMorris, K.L. / Buffalo, C.Z. / Ren, X. / Hurley, J.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 AI 120691 United States
CitationJournal: Cell / Year: 2018
Title: HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation.
Authors: Kyle L Morris / Cosmo Z Buffalo / Christina M Stürzel / Elena Heusinger / Frank Kirchhoff / Xuefeng Ren / James H Hurley /
Abstract: The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, ...The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 14, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 8, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
L: Bone marrow stromal antigen 2, Nef protein chimera
T: Bone marrow stromal antigen 2, Nef protein chimera
B: AP-1 complex subunit beta-1
C: ADP-ribosylation factor 1
G: AP-1 complex subunit gamma-1
H: ADP-ribosylation factor 1
M: AP-1 complex subunit mu-1
S: AP-1 complex subunit sigma-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)306,92712
Polymers305,8328
Non-polymers1,0954
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27460 Å2
ΔGint-129 kcal/mol
Surface area83650 Å2

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Components

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Protein , 2 types, 4 molecules LTCH

#1: Protein Bone marrow stromal antigen 2, Nef protein chimera / BST-2 / HM1.24 antigen / Tetherin / 3'ORF / Negative factor / F-protein


Mass: 29964.326 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Human immunodeficiency virus 1
Gene: BST2, nef / Production host: Escherichia coli (E. coli) / References: UniProt: Q10589, UniProt: Q90VU7
#3: Protein ADP-ribosylation factor 1 / ARF1


Mass: 22314.250 Da / Num. of mol.: 2 / Mutation: Q71L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARF1 / Production host: Escherichia coli (E. coli) / References: UniProt: P84077

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AP-1 complex subunit ... , 4 types, 4 molecules BGMS

#2: Protein AP-1 complex subunit beta-1 / AP-1 transcription factor / Adaptor protein complex AP-1 subunit beta-1 / Adaptor-related protein complex 1 subunit beta-1 / ...Adaptor protein complex AP-1 subunit beta-1 / Adaptor-related protein complex 1 subunit beta-1 / Beta-1-adaptin / Beta-adaptin 1 / Clathrin assembly protein complex 1 beta large chain / Golgi adaptor HA1/AP1 adaptin beta subunit


Mass: 66152.547 Da / Num. of mol.: 1 / Mutation: K359R, E476K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP1B1, ADTB1, BAM22, CLAPB2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q10567
#4: Protein AP-1 complex subunit gamma-1 / AP-1 transcription factor / Adaptor protein complex AP-1 subunit gamma-1 / Adaptor-related protein complex 1 subunit gamma-1 / ...Adaptor protein complex AP-1 subunit gamma-1 / Adaptor-related protein complex 1 subunit gamma-1 / Clathrin assembly protein complex 1 gamma-1 large chain / Gamma-adaptin / Gamma1-adaptin / Golgi adaptor HA1/AP1 adaptin subunit gamma-1


Mass: 68194.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap1g1, Adtg, Clapg1 / Production host: Escherichia coli (E. coli) / References: UniProt: P22892
#5: Protein AP-1 complex subunit mu-1 / AP-1 transcription factor / AP-mu chain family member mu1A / Adaptor protein complex AP-1 subunit mu-1 / Adaptor-related ...AP-mu chain family member mu1A / Adaptor protein complex AP-1 subunit mu-1 / Adaptor-related protein complex 1 subunit mu-1 / Clathrin assembly protein complex 1 mu-1 medium chain 1 / Clathrin coat assembly protein AP47 / Clathrin coat-associated protein AP47 / Golgi adaptor HA1/AP1 adaptin mu-1 subunit / Mu-adaptin 1 / Mu1A-adaptin


Mass: 48606.730 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap1m1, Cltnm / Production host: Escherichia coli (E. coli) / References: UniProt: P35585
#6: Protein AP-1 complex subunit sigma-3 / AP-1 transcription factor / Adaptor protein complex AP-1 subunit sigma-1C / Adaptor-related protein complex 1 subunit sigma-1C ...Adaptor protein complex AP-1 subunit sigma-1C / Adaptor-related protein complex 1 subunit sigma-1C / Clathrin assembly protein complex 1 sigma-1C small chain / Golgi adaptor HA1/AP1 adaptin sigma-1C subunit / Sigma 1C subunit of AP-1 clathrin / Sigma-adaptin 1C / Sigma1C-adaptin


Mass: 18321.338 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP1S3 / Production host: Escherichia coli (E. coli) / References: UniProt: Q96PC3

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Non-polymers , 2 types, 4 molecules

#7: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1AP-1:Arf1:tetherin-NefCOMPLEX1,2,3,4,5,60RECOMBINANT
2AP-1 sigma-3:Arf1:tetherin-NefCOMPLEX1,2,3,61RECOMBINANT
3AP-1 mu-1 and gamma-1COMPLEX4,51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 294 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 46849 X / Nominal defocus max: 2000 nm / Nominal defocus min: 750 nm / Cs: 2.6 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9.5 sec. / Electron dose: 62.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2200
Image scansWidth: 3712 / Height: 3840 / Movie frames/image: 38 / Used frames/image: 3-28

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatch0.5particle selection
4Gctf1.06CTF correction
7UCSF Chimera1.12model fitting
9RELIONinitial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIX1.13model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 252212
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53123 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient / Details: Phenix real space refine
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-IDPdb chain residue range
14P6Z1
24HMYC1
31O3YA11001-1001
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00516879
ELECTRON MICROSCOPYf_angle_d0.82522835
ELECTRON MICROSCOPYf_dihedral_angle_d8.40510352
ELECTRON MICROSCOPYf_chiral_restr0.0532636
ELECTRON MICROSCOPYf_plane_restr0.0062890

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