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- EMDB-7456: Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer... -

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Basic information

Entry
Database: EMDB / ID: EMD-7456
TitleStructure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit after focussed classification
Map data
SampleClosed trimer assembly of AP-1:Arf1:Tetherin-Nef
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.05 Å
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.
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: 0.0323
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0323
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_7456.map.gz / Format: CCP4 / Size: 42.9 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 224 pix.
= 239.008 Å
1.07 Å/pix.
x 224 pix.
= 239.008 Å
1.07 Å/pix.
x 224 pix.
= 239.008 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: 0.0323 / Movie #1: 0.0323
Minimum - Maximum-0.0566386 - 0.10795069
Average (Standard dev.)0.00056594145 (±0.004608694)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 239.00801 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0671.0671.067
M x/y/z224224224
origin x/y/z0.0060.0060.006
length x/y/z239.008239.008239.008
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ208208208
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.1270.1800.001

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Supplemental data

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Sample components

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Entire Closed trimer assembly of AP-1:Arf1:Tetherin-Nef

EntireName: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef / Number of components: 1

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Component #1: protein, Closed trimer assembly of AP-1:Arf1:Tetherin-Nef

ProteinName: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef / Recombinant expression: No
MassTheoretical: 230 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: 62.4 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 - 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: 2200

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 26684
3D reconstructionSoftware: RELION / Resolution: 4.05 Å / Resolution method: FSC 0.143 CUT-OFF

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