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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 ...) x 4
  • ADP-ribosylation factor 1
  • 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 plasmacytoid dendritic cell cytokine production / negative regulation of intracellular transport of viral material / response to interferon-beta / basolateral protein secretion / endosome to melanosome transport / AP-1 adaptor complex / Lysosome Vesicle Biogenesis / mitotic cleavage furrow ingression / trans-Golgi Network Vesicle Budding / protein trimerization ...negative regulation of plasmacytoid dendritic cell cytokine production / negative regulation of intracellular transport of viral material / response to interferon-beta / basolateral protein secretion / endosome to melanosome transport / AP-1 adaptor complex / Lysosome Vesicle Biogenesis / mitotic cleavage furrow ingression / trans-Golgi Network Vesicle Budding / protein trimerization / response to interferon-alpha / platelet dense granule organization / negative regulation of glycoprotein biosynthetic process / Glycosphingolipid transport / metalloendopeptidase inhibitor activity / 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 / positive regulation of leukocyte proliferation / 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 / COPI-dependent Golgi-to-ER retrograde traffic / azurophil granule membrane / clathrin binding / negative regulation of viral genome replication / Lysosome Vesicle Biogenesis / Golgi Associated Vesicle Biogenesis / Synthesis of PIPs at the plasma membrane / cell leading edge / B cell activation / MHC class I protein binding / host cell Golgi membrane / intracellular copper ion homeostasis / response to type II interferon / protein targeting / side of membrane / COPI-mediated anterograde transport / vesicle-mediated transport / clathrin-coated pit / viral life cycle / regulation of calcium-mediated signaling / multivesicular body / Neutrophil degranulation / MHC class II antigen presentation / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / cytoplasmic vesicle membrane / negative regulation of cell migration / Nef mediated downregulation of MHC class I complex cell surface expression / sarcomere / trans-Golgi network membrane / small monomeric GTPase / regulation of actin cytoskeleton organization / intracellular protein transport / kidney development / trans-Golgi network / negative regulation of cell growth / cellular response to virus / SH3 domain binding / virion component / response to virus / SARS-CoV-1 activates/modulates innate immune responses / Interferon alpha/beta signaling / synaptic vesicle / presynapse / heart development / ATPase binding / defense response to virus / early endosome / positive regulation of canonical NF-kappaB signal transduction / neuron projection / postsynaptic density / symbiont-mediated suppression of host innate immune response / apical plasma membrane / membrane raft / Golgi membrane / signaling receptor binding / protein domain specific binding / lysosomal membrane / innate immune response / focal adhesion / intracellular membrane-bounded organelle / GTPase activity / synapse / Neutrophil degranulation / protein kinase binding / GTP binding
Similarity search - Function
Bone marrow stromal antigen 2 / Bone marrow stromal antigen 2 / Beta-Lactamase - #60 / 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 ...Bone marrow stromal antigen 2 / Bone marrow stromal antigen 2 / Beta-Lactamase - #60 / 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 / Leucine-rich Repeat Variant / Leucine-rich Repeat Variant / Beta-Lactamase / Rab subfamily of small GTPases / Armadillo-like helical / Alpha Horseshoe / Small GTP-binding protein domain / Armadillo-type fold / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / AP-1 complex subunit gamma-1 / AP-1 complex subunit mu-1 / ADP-ribosylation factor 1 / AP-1 complex subunit beta-1 / Bone marrow stromal antigen 2 / Protein Nef / AP-1 complex subunit sigma-3
Similarity search - Component
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.
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
Revision 1.2Mar 13, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

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  • Deposited structure unit
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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
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, microscopy
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


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 / 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 / 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-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 / 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


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


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-NefCOMPLEX#1-#60RECOMBINANT
2AP-1 sigma-3:Arf1:tetherin-NefCOMPLEX#1-#3, #61RECOMBINANT
3AP-1 mu-1 and gamma-1COMPLEX#4-#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 FIELD / 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 / Specimen holder 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

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
14P6Z

4p6z

4P6Z1
24HMY

4hmy

C4HMY2
31O3Y

1o3y

A1O3Y31001-1001
Refine LS restraints
Refine-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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