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- PDB-6cm9: Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer... -
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Basic information
Entry | Database: PDB / ID: 6cm9 | ||||||
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Title | Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit | ||||||
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![]() | VIRAL PROTEIN / PROTEIN TRANSPORT / AP / HIV / Nef / trafficking | ||||||
Function / homology | ![]() negative regulation of plasmacytoid dendritic cell cytokine production / negative regulation of intracellular transport of viral material / response to interferon-beta / basolateral protein secretion / perturbation by virus of host immune response / negative regulation of CD4 production / mitotic cleavage furrow ingression / endosome to melanosome transport / trans-Golgi Network Vesicle Budding / AP-1 adaptor complex ...negative regulation of plasmacytoid dendritic cell cytokine production / negative regulation of intracellular transport of viral material / response to interferon-beta / basolateral protein secretion / perturbation by virus of host immune response / negative regulation of CD4 production / mitotic cleavage furrow ingression / endosome to melanosome transport / trans-Golgi Network Vesicle Budding / AP-1 adaptor complex / Lysosome Vesicle Biogenesis / platelet dense granule organization / protein trimerization / Glycosphingolipid transport / regulation of receptor internalization / response to interferon-alpha / 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 / Intra-Golgi traffic / metalloendopeptidase inhibitor activity / 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 / symbiont-mediated suppression of host apoptosis / clathrin adaptor activity / suppression by virus of host autophagy / MHC class II antigen presentation / positive regulation of leukocyte proliferation / CD4 receptor binding / Nef Mediated CD4 Down-regulation / thioesterase binding / dendritic spine organization / determination of left/right symmetry / long-term synaptic depression / clathrin-coated vesicle / COPI-dependent Golgi-to-ER retrograde traffic / Lysosome Vesicle Biogenesis / azurophil granule membrane / clathrin binding / negative regulation of viral genome replication / Golgi Associated Vesicle Biogenesis / B cell activation / response to type II interferon / cell leading edge / MHC class I protein binding / host cell Golgi membrane / Synthesis of PIPs at the plasma membrane / intracellular copper ion homeostasis / protein targeting / side of membrane / COPI-mediated anterograde transport / clathrin-coated pit / regulation of calcium-mediated signaling / vesicle-mediated transport / viral life cycle / MHC class II antigen presentation / Neutrophil degranulation / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / multivesicular body / sarcomere / small monomeric GTPase / trans-Golgi network membrane / negative regulation of cell migration / Nef mediated downregulation of MHC class I complex cell surface expression / kidney development / virion component / regulation of actin cytoskeleton organization / intracellular protein transport / response to virus / trans-Golgi network / cytoplasmic vesicle membrane / negative regulation of cell growth / cellular response to virus / SH3 domain binding / SARS-CoV-1 activates/modulates innate immune responses / Interferon alpha/beta signaling / presynapse / heart development / ATPase binding / positive regulation of canonical NF-kappaB signal transduction / defense response to virus / postsynaptic density / early endosome / neuron projection / apical plasma membrane / membrane raft / protein domain specific binding / Golgi membrane / lysosomal membrane / intracellular membrane-bounded organelle / innate immune response / signaling receptor binding / focal adhesion / GTPase activity / synapse / Neutrophil degranulation / GTP binding / apoptotic process Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.73 Å | ||||||
![]() | Morris, K.L. / Buffalo, C.Z. / Ren, X. / Hurley, J.H. | ||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 415.3 KB | Display | ![]() |
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PDB format | ![]() | 323.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 7457MC ![]() 7453C ![]() 7454C ![]() 7455C ![]() 7456C ![]() 7458C ![]() 7563C ![]() 6criC ![]() 6d83C ![]() 6d84C ![]() 6dffC M: map data used to model this data C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 115.4 Data #1: Dose weighted particle stack of AP1:Arf1:tetherin-HIV-Nef [picked particles - multiframe - unprocessed]) ![]() Data size: 34.0 Data #1: Dose weighted particle stack of AP1:Arf1:tetherin-HIV-Nef closed trimer [picked particles - multiframe - unprocessed]) ![]() Data size: 10.1 Data #1: Dose weight particle stack of AP1:Arf1:tetherin-HIV-Nef closed trimer after monomeric subunit extraction using LocalRec [picked particles - multiframe - processed]) |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 2 types, 5 molecules LTNCH
#1: Protein | Mass: 29964.326 Da / Num. of mol.: 3 Fragment: Tetherin UNP residues 2-21, Nef UNP residues 1-206 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Gene: BST2, nef / Production host: ![]() ![]() #3: Protein | Mass: 22314.250 Da / Num. of mol.: 2 / Fragment: UNP residues 17-181 / Mutation: Q71L Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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-AP-1 complex subunit ... , 4 types, 4 molecules BGMS
#2: Protein | Mass: 66152.547 Da / Num. of mol.: 1 / Mutation: K359R, E476K Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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#4: Protein | Mass: 68194.094 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#5: Protein | Mass: 48606.730 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#6: Protein | Mass: 18321.338 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
-Non-polymers , 2 types, 4 molecules ![](data/chem/img/GTP.gif)
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#7: Chemical | #8: Chemical | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Structure of the HIV-Nef tetherin cargo bound AP-1:Arf1 closed trimer monomeric subunit Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT |
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Molecular weight | Value: 0.7 MDa / Experimental value: YES |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 8 |
Specimen | Conc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3 |
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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 scans | Width: 3712 / Height: 3840 / Movie frames/image: 38 / Used frames/image: 3-38 |
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Processing
Software | Name: PHENIX / Version: 1.13_2998: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 252212 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53841 / Algorithm: BACK PROJECTION / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: 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
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Refine LS restraints |
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