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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-6389 | |||||||||
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Title | Electron cryo-microscopy of AP-1:Arf1:Nef open trimer | |||||||||
![]() | Reconstruction of Ap-1:Arf1:Nef open trimer | |||||||||
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![]() | adaptor protein-1 / clathrin cage formation | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 17.0 Å | |||||||||
![]() | Shen QT / Ren X / Zhang R / Lee H / Hurley J | |||||||||
![]() | ![]() Title: HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons. Authors: Qing-Tao Shen / Xuefeng Ren / Rui Zhang / Il-Hyung Lee / James H Hurley / ![]() Abstract: The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and ...The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat. | |||||||||
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 | ![]() | 7.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.6 KB 11.6 KB | Display Display | ![]() |
Images | ![]() | 105 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 78.7 KB | Display | ![]() |
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Full document | ![]() | 77.9 KB | Display | |
Data in XML | ![]() | 495 B | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Reconstruction of Ap-1:Arf1:Nef open trimer | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.64 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : AP-1:Arf1:Nef complex
Entire | Name: AP-1:Arf1:Nef complex |
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Components |
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-Supramolecule #1000: AP-1:Arf1:Nef complex
Supramolecule | Name: AP-1:Arf1:Nef complex / type: sample / ID: 1000 / Oligomeric state: one AP-1 binds to two Arf1 and one Nef / Number unique components: 3 |
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Molecular weight | Experimental: 850 KDa / Theoretical: 870 KDa / Method: multi-angle light scattering |
-Macromolecule #1: adaptor protein-1
Macromolecule | Name: adaptor protein-1 / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #2: Arf1
Macromolecule | Name: Arf1 / type: protein_or_peptide / ID: 2 / Name.synonym: ADP-ribosylation factor-1 / Number of copies: 2 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 200 KDa / Theoretical: 200 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #3: Nef
Macromolecule | Name: Nef / type: protein_or_peptide / ID: 3 / Name.synonym: negative factor / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 290 KDa / Theoretical: 290 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.2 mg/mL |
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Buffer | pH: 8 / Details: 20 mM Tris, 200 mM NaCl, 0.3 mM TCEP |
Grid | Details: 400 mesh copper grid with thin carbon support |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2 seconds before plunging |
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Electron microscopy
Microscope | FEI TITAN |
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Date | Mar 30, 2015 |
Image recording | Category: CCD / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Number real images: 1000 / Average electron dose: 45.9 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 80000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 80000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
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Image processing
Details | The particles were selected using the DoGrun software package. |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 56000 |
Final two d classification | Number classes: 10 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: ![]() |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |