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- EMDB-7563: Structure of the cargo bound AP-1:Arf1:tetherin-Nef stable closed... -

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Entry
Database: EMDB / ID: 7563
TitleStructure of the cargo bound AP-1:Arf1:tetherin-Nef stable closed trimer
Map dataHIV-Nef tetherin cargo bound AP-1:Arf1 stable closed trimer
SampleStructure of the cargo bound AP-1:Arf1:tetherin-Nef stable closed trimer
  • Bone marrow stromal antigen 2, Protein Nef chimera
  • (AP-1 complex subunit ...AP-1 transcription factor) x 4
  • ADP-ribosylation factor 1ARF1
  • (ligand) x 2
Function / homologyClathrin adaptor complexes small chain signature. / Clathrin derived vesicle budding / Beta-adaptin appendage, C-terminal subdomain / Neutrophil degranulation / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / TBP domain superfamily / Golgi Associated Vesicle Biogenesis / Armadillo-like helical / Longin-like domain superfamily ...Clathrin adaptor complexes small chain signature. / Clathrin derived vesicle budding / Beta-adaptin appendage, C-terminal subdomain / Neutrophil degranulation / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / TBP domain superfamily / Golgi Associated Vesicle Biogenesis / Armadillo-like helical / Longin-like domain superfamily / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Small GTPase superfamily, ARF/SAR type / Small GTP-binding protein domain / Lysosome Vesicle Biogenesis / MHC class II antigen presentation / AP-1/2/4 complex subunit beta / Clathrin/coatomer adaptor, adaptin-like, N-terminal / HIV negative factor Nef / Clathrin adaptor, mu subunit / Clathrin adaptor complex, small chain / Armadillo / Nef Mediated CD4 Down-regulation / Clathrin adaptor complexes medium chain signature 2. / Clathrin adaptor complexes medium chain signature 1. / Synthesis of PIPs at the Golgi membrane / Synthesis of PIPs at the plasma membrane / Gamma-adaptin ear (GAE) domain profile. / Mu homology domain (MHD) profile. / Nef mediated downregulation of MHC class I complex cell surface expression / Armadillo-type fold / Gamma-adaptin ear (GAE) domain / Adaptor protein complex, sigma subunit / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / Neutrophil degranulation / Golgi Associated Vesicle Biogenesis / Bone marrow stromal antigen 2 / Lysosome Vesicle Biogenesis / Beta2-adaptin appendage, C-terminal sub-domain / Adaptin C-terminal domain / MHC class II antigen presentation / Adaptin N terminal region / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / Negative factor, (F-Protein) or Nef / Interferon alpha/beta signaling / ADP-ribosylation factor family / small GTPase Arf family profile. / Intra-Golgi traffic / Mu homology domain / AP complex subunit beta / Bone marrow stromal antigen 2 / P-loop containing nucleoside triphosphate hydrolase / HIV-1 Nef protein, anchor domain superfamily / HIV-1 Nef protein, core domain superfamily / in:ipr024156: / AP complex, mu/sigma subunit / Clathrin adaptor, mu subunit, conserved site / COPI-mediated anterograde transport / Adaptor protein complex AP-1, gamma subunit / AP-2 complex subunit mu, C-terminal superfamily / COPI-dependent Golgi-to-ER retrograde traffic / lysosomal membrane organization / negative regulation of plasmacytoid dendritic cell cytokine production / Golgi to transport vesicle transport / synaptic vesicle budding / phospholipase D activator activity / positive regulation of late endosome to lysosome transport / regulation of phospholipid metabolic process / negative regulation of intracellular transport of viral material / positive regulation of natural killer cell degranulation / negative regulation of CD4 biosynthetic process / negative regulation by symbiont of host T-cell mediated immune response / mitotic cleavage furrow ingression / AP-1 adaptor complex / endosome to melanosome transport / positive regulation of ER to Golgi vesicle-mediated transport / regulation of Arp2/3 complex-mediated actin nucleation / Golgi to lysosome transport / very-low-density lipoprotein particle assembly / response to interferon-beta / melanosome organization / suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I / COPI-coated vesicle / regulation of receptor internalization / clathrin adaptor complex / response to interferon-alpha / dendritic spine organization / membrane coat / metalloendopeptidase inhibitor activity / positive regulation of natural killer cell mediated cytotoxicity / positive regulation of calcium ion-dependent exocytosis / GTP-dependent protein binding / long-term synaptic depression / determination of left/right symmetry / regulation of calcium-mediated signaling / thioesterase binding / azurophil granule membrane / CD4 receptor binding / peroxisomal membrane
Function and homology information
SourceHomo sapiens (human) / Human immunodeficiency virus 1 / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / 6.8 Å resolution
AuthorsMorris KL / Buffalo CZ / 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: 6cri

SummaryFull reportAbout validation report
DateDeposition: Mar 18, 2018 / Header (metadata) release: Apr 11, 2018 / Map release: Aug 1, 2018 / Last update: Aug 8, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0149
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0149
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6cri
  • Surface level: 0.0149
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7563.map.gz (map file in CCP4 format, 226493 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
384 pix
1.07 Å/pix.
= 409.728 Å
384 pix
1.07 Å/pix.
= 409.728 Å
384 pix
1.07 Å/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 Level:0.0149 (by author), 0.0149 (movie #1):
Minimum - Maximum-0.017942984 - 0.058546383
Average (Standard dev.)0.0003145799 (0.002489533)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions384384384
Origin0.0.0.
Limit383.383.383.
Spacing384384384
CellA=B=C: 409.72803 Å
α=β=γ: 90.0 deg.

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/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0180.0590.000

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Supplemental data

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Sample components

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Entire Structure of the cargo bound AP-1:Arf1:tetherin-Nef stable closed...

EntireName: Structure of the cargo bound AP-1:Arf1:tetherin-Nef stable closed trimer
Number of components: 9

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Component #1: protein, Structure of the cargo bound AP-1:Arf1:tetherin-Nef stab...

ProteinName: Structure of the cargo bound AP-1:Arf1:tetherin-Nef stable closed trimer
Recombinant expression: No
MassTheoretical: 700 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Bone marrow stromal antigen 2, Protein Nef chimera

ProteinName: Bone marrow stromal antigen 2, Protein Nef chimera / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 29.964326 kDa
SourceSpecies: Human immunodeficiency virus 1
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, AP-1 complex subunit beta-1

ProteinName: AP-1 complex subunit beta-1AP-1 transcription factor / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 64.458656 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, ADP-ribosylation factor 1

ProteinName: ADP-ribosylation factor 1ARF1 / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 18.9366 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, AP-1 complex subunit gamma-1

ProteinName: AP-1 complex subunit gamma-1AP-1 transcription factor / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 66.401055 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, AP-1 complex subunit mu-1

ProteinName: AP-1 complex subunit mu-1AP-1 transcription factor / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 48.475535 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: protein, AP-1 complex subunit sigma-3

ProteinName: AP-1 complex subunit sigma-3AP-1 transcription factor / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 16.99885 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: ligand, GUANOSINE-5'-TRIPHOSPHATE

LigandName: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 0.52318 kDa

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Component #9: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.07 mg/ml
Buffer solution: 20 mM Tris at pH 8.0, 200 mM NaCl, 5 mM MgCl2, and 0.5 mM TCEP
pH: 8
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 296 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: 62.4 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 11108
3D reconstructionResolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: Correlation coefficient / Refinement space: REAL / Overall bvalue: 425
Output model

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