|Entry||Database: EMDB / ID: 7456|
|Title||Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit after focussed classification|
|Map data||Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit after focussed classification|
|Sample||Closed trimer assembly of AP-1:Arf1:Tetherin-Nef:|
|Source||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / 4.05 Å resolution|
|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.
|Date||Deposition: Feb 2, 2018 / Header (metadata) release: Mar 14, 2018 / Map release: Jul 25, 2018 / Last update: Aug 8, 2018|
|Structure viewer||EM map: |
Downloads & links
|File||emd_7456.map.gz (map file in CCP4 format, 44958 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.067 Å|
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: 230 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. X (nominal), 46849. X (calibrated) / Cs: 2.6 mm / Imaging mode: BRIGHT FIELD / Defocus: 750.0 - nm|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 2200|
|Raw data||EMPIAR-10176 (Title: Single particle cryo-EM dataset of the flexible and variable oligomeric state complex AP-1:Arf1:tetherin-HIV-Nef|
Data size: 115.4
Data #1: Dose weighted particle stack of AP1:Arf1:tetherin-HIV-Nef [picked particles - multiframe - unprocessed])
EMPIAR-10177 (Title: Single particle cryo-EM dataset of the flexible and variable oligomeric state complex AP-1:Arf1:tetherin-HIV-Nef
Data size: 34.0
Data #1: Dose weighted particle stack of AP1:Arf1:tetherin-HIV-Nef closed trimer [picked particles - multiframe - unprocessed])
EMPIAR-10178 (Title: Single particle cryo-EM dataset of the flexible and variable oligomeric state complex AP-1:Arf1:tetherin-HIV-Nef
Data size: 10.1
Data #1: Dose weight particle stack of AP1:Arf1:tetherin-HIV-Nef closed trimer after monomeric subunit extraction using LocalRec [picked particles - multiframe - processed])
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 26684|
|3D reconstruction||Resolution: 4.05 Å / Resolution method: FSC 0.143 CUT-OFF|
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