|Entry||Database: EMDB / ID: EMD-7458|
|Title||Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer|
|Sample||Closed trimer assembly of AP-1:Arf1:Tetherin-Nef|
|Biological species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 4.27 Å|
|Authors||Morris KL / Buffalo CZ / Hurley JH|
|Citation||Journal: 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.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_7458.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.067 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Closed trimer assembly of AP-1:Arf1:Tetherin-Nef
|Entire||Name: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef / Number of components: 1|
-Component #1: protein, Closed trimer assembly of AP-1:Arf1:Tetherin-Nef
|Protein||Name: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef / Recombinant expression: No|
|Mass||Theoretical: 700 kDa|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen 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
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 294 K / Humidity: 100 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 62.4 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 22500.0 X (nominal), 46849.0 X (calibrated) / Cs: 2.6 mm / Imaging mode: BRIGHT FIELD / Defocus: 750.0 - 2000.0 nm|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 2200|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 61929|
|3D reconstruction||Software: RELION / Resolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF|
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