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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 dataStructure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit after focussed classification
Sample
  • Complex: Closed 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
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesR01 AI 120691 United States
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-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing 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
  • Download
  • 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

emd_7456.png

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)
AnnotationStructure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit after focussed classification
Voxel sizeX=Y=Z: 1.067 Å
Density
Contour LevelBy AUTHOR: 0.0323 / Movie #1: 0.0323
Minimum - Maximum-0.12749371 - 0.18036003
Average (Standard dev.)0.00056594145 (±0.0060708364)
SymmetrySpace group: 0
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/NZ320320320
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
Components
  • Complex: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef

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Supramolecule #1: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef

SupramoleculeName: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 230 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 8
Details: 20 mM Tris at pH 8.0, 200 mM NaCl, 5 mM MgCl2, and 0.5 mM TCEP
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 294 K / Instrument: FEI VITROBOT MARK IV
DetailsAP-1:Arf1:Tetherin-Nef

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 46849 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.6 mm / Nominal defocus min: 0.75 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3712 pixel / Digitization - Dimensions - Height: 3840 pixel / Digitization - Frames/image: 3-38 / Number grids imaged: 1 / Number real images: 2200 / Average exposure time: 9.5 sec. / Average electron dose: 62.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 252212
CTF correctionSoftware - Name: RELION
Startup modelType of model: OTHER / Details: Relion localized reconstruction (Ilca 2015)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 26684

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