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- EMDB-7454: Structure of the HIV-Nef dileucine mutant bound AP-1:Arf1 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-7454
TitleStructure of the HIV-Nef dileucine mutant bound AP-1:Arf1 dimer
Map data
SampleDimeric assembly of AP-1:Arf1:Tetherin-Nef dileucine (LL164-165AA) mutant
Function / homology
Function and homology information


negative regulation of intracellular transport of viral material / negative regulation of plasmacytoid dendritic cell cytokine production / phospholipase D activator activity / mitotic cleavage furrow ingression / Golgi to transport vesicle transport / synaptic vesicle budding / lysosomal membrane organization / positive regulation of late endosome to lysosome transport / regulation of phospholipid metabolic process / negative regulation of CD4 biosynthetic process ...negative regulation of intracellular transport of viral material / negative regulation of plasmacytoid dendritic cell cytokine production / phospholipase D activator activity / mitotic cleavage furrow ingression / Golgi to transport vesicle transport / synaptic vesicle budding / lysosomal membrane organization / positive regulation of late endosome to lysosome transport / regulation of phospholipid metabolic process / 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 / dendritic spine organization / GTP-dependent protein binding / positive regulation of natural killer cell mediated cytotoxicity / long-term synaptic depression / CD4 receptor binding / determination of left/right symmetry / thioesterase binding / regulation of calcium-mediated signaling / azurophil granule membrane / peroxisomal membrane / host cell Golgi membrane / clathrin binding / positive regulation of endocytosis / positive regulation of dendritic spine development / clathrin-coated vesicle / kinesin binding / response to interferon-gamma / MHC class I protein binding / 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 / trans-Golgi network membrane / protein targeting / clathrin-coated pit / Rab GTPase binding / clathrin-coated vesicle membrane / cellular response to virus / antigen processing and presentation of exogenous peptide antigen via MHC class II / anchored component of membrane / regulation of actin cytoskeleton organization / negative regulation of cell migration / positive regulation of protein secretion / sarcomere / actin filament organization / multivesicular body / vesicle-mediated transport / collagen binding / microtubule cytoskeleton organization / interleukin-12-mediated signaling pathway / response to virus / microtubule cytoskeleton / GDP binding / recycling endosome / cytoplasmic vesicle membrane / type I interferon signaling pathway / intracellular protein transport / trans-Golgi network / virion / regulation of defense response to virus by virus / viral life cycle / SH3 domain binding / negative regulation of cell growth / lysosomal membrane / late endosome / defense response to virus / apical plasma membrane / heart development / ATPase binding / positive regulation of I-kappaB kinase/NF-kappaB signaling / protein C-terminus binding / postsynaptic density / GTPase activity / neuron projection / Golgi membrane
Clathrin adaptor, mu subunit / AP complex, mu/sigma subunit / Beta-adaptin appendage, C-terminal subdomain / Clathrin adaptor, appendage, Ig-like subdomain superfamily / HIV negative factor Nef / Clathrin adaptor complex, small chain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / TBP domain superfamily / Armadillo-like helical ...Clathrin adaptor, mu subunit / AP complex, mu/sigma subunit / Beta-adaptin appendage, C-terminal subdomain / Clathrin adaptor, appendage, Ig-like subdomain superfamily / HIV negative factor Nef / Clathrin adaptor complex, small chain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / TBP domain superfamily / Armadillo-like helical / Adaptor protein complex, sigma subunit / Small GTP-binding protein domain / Adaptor protein complex AP-1, gamma subunit / Clathrin adaptor, mu subunit, conserved site / Small GTPase superfamily, ARF/SAR type / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / AP-1/2/4 complex subunit beta / Armadillo / Gamma-adaptin ear (GAE) domain / Bone marrow stromal antigen 2 / AP complex subunit beta / P-loop containing nucleoside triphosphate hydrolase / HIV-1 Nef protein, anchor domain superfamily / HIV-1 Nef protein, core domain superfamily / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Mu homology domain / Longin-like domain superfamily / AP-2 complex subunit mu, C-terminal superfamily / Armadillo-type fold
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
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.72 Å
AuthorsBuffalo CZ / Morris KL / Hurley JH
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 ReportPDB-ID: 6d84

SummaryFull reportAbout validation report
History
DepositionFeb 2, 2018-
Header (metadata) releaseMar 14, 2018-
Map releaseJul 25, 2018-
UpdateMay 15, 2019-
Current statusMay 15, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0184
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0184
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6d84
  • Surface level: 0.0184
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_7454.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 384 pix.
= 409.728 Å
1.07 Å/pix.
x 384 pix.
= 409.728 Å
1.07 Å/pix.
x 384 pix.
= 409.728 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.067 Å
Density
Contour LevelBy AUTHOR: 0.0184 / Movie #1: 0.0184
Minimum - Maximum-0.035247605 - 0.043077793
Average (Standard dev.)0.0006186019 (±0.003222912)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.152 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0671.0671.067
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z409.728409.728409.728
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ208208208
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1180.1070.000

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Supplemental data

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Sample components

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Entire Dimeric assembly of AP-1:Arf1:Tetherin-Nef dileucine (LL164-165AA...

EntireName: Dimeric assembly of AP-1:Arf1:Tetherin-Nef dileucine (LL164-165AA) mutant
Number of components: 1

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Component #1: protein, Dimeric assembly of AP-1:Arf1:Tetherin-Nef dileucine (LL...

ProteinName: Dimeric assembly of AP-1:Arf1:Tetherin-Nef dileucine (LL164-165AA) mutant
Recombinant expression: No
MassTheoretical: 470 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.7 mg/mL
Buffer solution: 20 mM Tris at pH 8.0, 200 mM NaCl, 5 mM MgCl2, and 0.5 mM TCEP
pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 294 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 39.7 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500.0 X (nominal), 46849.0 X (calibrated) / Cs: 2.6 mm / Imaging mode: BRIGHT FIELD / Defocus: 750.0 - 2500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1989

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 42902
3D reconstructionSoftware: RELION / Resolution: 6.72 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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