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- EMDB-7456: Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-7456 | |||||||||
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Title | Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit after focussed classification | |||||||||
![]() | Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit after focussed classification | |||||||||
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Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.05 Å | |||||||||
![]() | Morris KL / Buffalo CZ / Hurley JH | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 5.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.4 KB 12.4 KB | Display Display | ![]() |
Images | ![]() | 680.4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7453C ![]() 7454C ![]() 7455C ![]() 7457C ![]() 7458C ![]() 7563C ![]() 6cm9C ![]() 6criC ![]() 6d83C ![]() 6d84C ![]() 6dffC C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 115.4 Data #1: Dose weighted particle stack of AP1:Arf1:tetherin-HIV-Nef [picked particles - multiframe - unprocessed]) ![]() Data size: 34.0 Data #1: Dose weighted particle stack of AP1:Arf1:tetherin-HIV-Nef closed trimer [picked particles - multiframe - unprocessed]) ![]() 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]) |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Structure of the cargo bound AP-1:Arf1:tetherin-Nef closed trimer monomeric subunit after focussed classification | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.067 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 0 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Closed trimer assembly of AP-1:Arf1:Tetherin-Nef
Entire | Name: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef |
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Components |
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-Supramolecule #1: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef
Supramolecule | Name: Closed trimer assembly of AP-1:Arf1:Tetherin-Nef / type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Molecular weight | Theoretical: 230 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.7 mg/mL |
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Buffer | pH: 8 Details: 20 mM Tris at pH 8.0, 200 mM NaCl, 5 mM MgCl2, and 0.5 mM TCEP |
Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 294 K / Instrument: FEI VITROBOT MARK IV |
Details | AP-1:Arf1:Tetherin-Nef |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 46849 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.6 mm / Nominal defocus min: 0.75 µm / Nominal magnification: 22500 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |