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- PDB-6owt: Structure of SIVsmm Nef and SMM tetherin bound to the clathrin ad... -

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Basic information

Entry
Database: PDB / ID: 6owt
TitleStructure of SIVsmm Nef and SMM tetherin bound to the clathrin adaptor AP-2 complex
Components
  • AP-2 complex subunit alpha
  • AP-2 complex subunit beta
  • AP-2 complex subunit sigma
  • Adaptor protein complex AP-2, mu1
  • Tetherin,Protein Nef
KeywordsPROTEIN TRANSPORT / AP / HIV / Nef / trafficking / viral restriction factor
Function / homology
Function and homology information


AP-type membrane coat adaptor complex / Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / clathrin coat / postsynaptic endocytic zone / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance ...AP-type membrane coat adaptor complex / Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / clathrin coat / postsynaptic endocytic zone / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Retrograde neurotrophin signalling / Trafficking of GluR2-containing AMPA receptors / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / extrinsic component of presynaptic endocytic zone membrane / clathrin adaptor complex / cardiac septum development / metalloendopeptidase inhibitor activity / Recycling pathway of L1 / MHC class II antigen presentation / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / clathrin coat assembly / Cargo recognition for clathrin-mediated endocytosis / Recycling pathway of L1 / Clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / Cargo recognition for clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / membrane coat / vesicle budding from membrane / positive regulation of protein localization to membrane / clathrin-dependent endocytosis / MHC class II antigen presentation / signal sequence binding / coronary vasculature development / aorta development / ventricular septum development / Neutrophil degranulation / clathrin binding / low-density lipoprotein particle receptor binding / Trafficking of GluR2-containing AMPA receptors / positive regulation of endocytosis / positive regulation of receptor internalization / synaptic vesicle endocytosis / negative regulation of protein localization to plasma membrane / vesicle-mediated transport / protein serine/threonine kinase binding / phosphatidylinositol binding / secretory granule / kidney development / intracellular protein transport / cytoplasmic side of plasma membrane / kinase binding / disordered domain specific binding / synaptic vesicle / heart development / protein-containing complex assembly / cytoplasmic vesicle / defense response to virus / transmembrane transporter binding / postsynapse / protein domain specific binding / innate immune response / intracellular membrane-bounded organelle / synapse / lipid binding / protein-containing complex binding / protein kinase binding / GTP binding / host cell plasma membrane / glutamatergic synapse / cell surface / Golgi apparatus / mitochondrion / membrane / plasma membrane
Similarity search - Function
Bone marrow stromal antigen 2 / Bone marrow stromal antigen 2 / Beta-Lactamase - #60 / AP-2 complex subunit sigma / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Alpha adaptin AP2, C-terminal domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain ...Bone marrow stromal antigen 2 / Bone marrow stromal antigen 2 / Beta-Lactamase - #60 / AP-2 complex subunit sigma / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Alpha adaptin AP2, C-terminal domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / 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 / Adaptor protein complex, sigma subunit / HIV-1 Nef protein, core domain superfamily / Negative factor, (F-Protein) or Nef / : / Beta-adaptin appendage, C-terminal subdomain / Beta2-adaptin appendage, C-terminal sub-domain / 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 / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / AP-2 complex subunit mu, C-terminal superfamily / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Longin-like domain superfamily / TBP domain superfamily / Armadillo/beta-catenin-like repeats / Armadillo / Leucine-rich Repeat Variant / Leucine-rich Repeat Variant / Beta-Lactamase / Armadillo-like helical / Alpha Horseshoe / Armadillo-type fold / 2-Layer Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Tetherin variant Sm2 / AP-2 complex subunit alpha-2 / AP-2 complex subunit sigma / AP-2 complex subunit beta / AP-2 complex subunit mu / Protein Nef / AP-2 complex subunit mu / AP-2 complex subunit alpha
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Mus musculus (house mouse)
Cercocebus atys (sooty mangabey)
Simian immunodeficiency virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsBuffalo, C.Z. / Ren, X. / Hurley, J.H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)F32 GM125209 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI120691 United States
CitationJournal: Cell Host Microbe / Year: 2019
Title: Structural Basis for Tetherin Antagonism as a Barrier to Zoonotic Lentiviral Transmission.
Authors: Cosmo Z Buffalo / Christina M Stürzel / Elena Heusinger / Dorota Kmiec / Frank Kirchhoff / James H Hurley / Xuefeng Ren /
Abstract: Tetherin is a host defense factor that physically prevents virion release from the plasma membrane. The Nef accessory protein of simian immunodeficiency virus (SIV) engages the clathrin adaptor AP-2 ...Tetherin is a host defense factor that physically prevents virion release from the plasma membrane. The Nef accessory protein of simian immunodeficiency virus (SIV) engages the clathrin adaptor AP-2 to downregulate tetherin via its DIWK motif. As human tetherin lacks DIWK, antagonism of tetherin by Nef is a barrier to simian-human transmission of non-human primate lentiviruses. To determine the molecular basis for tetherin counteraction, we reconstituted the AP-2 complex with a simian tetherin and SIV Nef and determined its structure by cryoelectron microscopy (cryo-EM). Nef refolds the first α-helix of the β2 subunit of AP-2 to a β hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of most other Nef substrates, including MHC class I, CD3, and CD4 but overlaps with the site for the restriction factor SERINC5. This structure explains the dependence of SIVs on tetherin DIWK and consequent barrier to human transmission.
History
DepositionMay 10, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 25, 2019Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Nov 20, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
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 / _em_admin.last_update
Revision 1.3May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: AP-2 complex subunit alpha
B: AP-2 complex subunit beta
M: Adaptor protein complex AP-2, mu1
T: Tetherin,Protein Nef
N: Tetherin,Protein Nef
S: AP-2 complex subunit sigma


Theoretical massNumber of molelcules
Total (without water)267,9586
Polymers267,9586
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein AP-2 complex subunit alpha


Mass: 104286.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2a2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q66HM2, UniProt: P18484*PLUS
#2: Protein AP-2 complex subunit beta / AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta ...AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta / Beta-2-adaptin / Beta-adaptin / Clathrin assembly protein complex 2 beta large chain / Plasma membrane adaptor HA2/AP2 adaptin beta subunit


Mass: 66953.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2b1, Clapb1 / Production host: Escherichia coli (E. coli) / References: UniProt: P62944
#3: Protein Adaptor protein complex AP-2, mu1


Mass: 16161.563 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2m1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5FWI9, UniProt: P84091*PLUS
#4: Protein Tetherin,Protein Nef


Mass: 31772.352 Da / Num. of mol.: 2 / Mutation: C55A
Source method: isolated from a genetically manipulated source
Details: protein chimera of the cytoplasmic tail of sooty mangabey (smm) linked to the N-terminus of SIV smm tetherin
Source: (gene. exp.) Cercocebus atys (sooty mangabey), (gene. exp.) Simian immunodeficiency virus
Gene: BST2, nef / Production host: Escherichia coli (E. coli) / References: UniProt: C3VHQ5, UniProt: Q4JGV0
#5: Protein AP-2 complex subunit sigma / Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / ...Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / Clathrin assembly protein 2 sigma small chain / Clathrin coat assembly protein AP17 / Clathrin coat-associated protein AP17 / Plasma membrane adaptor AP-2 17 kDa protein / Sigma-adaptin 3b / Sigma2-adaptin


Mass: 17011.662 Da / Num. of mol.: 1 / Mutation: N97C, C99S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2s1, Ap17, Claps2 / Production host: Escherichia coli (E. coli) / References: UniProt: P62744
Has protein modificationY

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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

ComponentName: Structure of SIVsmm Nef and SMM tetherin bound to the clathrin adaptor AP-2 complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2036 MDa / Experimental value: YES
Source (natural)Organism: Rattus norvegicus (Norway rat)
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 supportDetails: 25mA / Grid type: C-flat-2/1
VitrificationCryogen name: ETHANE

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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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.16 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15_3459: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 159406 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
14NEE

4nee

14NEE1PDBexperimental model
22XA7

2xa7

12XA72PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311872
ELECTRON MICROSCOPYf_angle_d0.59916228
ELECTRON MICROSCOPYf_dihedral_angle_d10.1857127
ELECTRON MICROSCOPYf_chiral_restr0.0411969
ELECTRON MICROSCOPYf_plane_restr0.0062071

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