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- EMDB-6388: Electron cryo-microscopy of AP-1:Arf1:Nef complex at 7 Angstrom r... -

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Basic information

Entry
Database: EMDB / ID: EMD-6388
TitleElectron cryo-microscopy of AP-1:Arf1:Nef complex at 7 Angstrom resolution
Map dataOne subunit of AP-1:Arf1:Nef complex
Sample
  • Sample: AP-1:Arf1:Nef
  • Protein or peptide: adaptor protein-1
  • Protein or peptide: Arf1
  • Protein or peptide: Nef
Keywordsadaptor protein-1
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsShen QT / Ren X / Zhang R / Lee H / Hurley J
CitationJournal: Science / Year: 2015
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
DepositionJul 17, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseNov 4, 2015-
UpdateNov 4, 2015-
Current statusNov 4, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.236
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.236
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6388.jpg

Downloads & links

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Map

FileDownload / File: emd_6388.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOne subunit of AP-1:Arf1:Nef complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.64 Å/pix.
x 128 pix.
= 337.92 Å		2.64 Å/pix.
x 128 pix.
= 337.92 Å		2.64 Å/pix.
x 128 pix.
= 337.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.64 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.236 / Movie #1: 0.236
Minimum - Maximum-0.67300588 - 1.06478286
Average (Standard dev.)-0.00035372 (±0.04631491)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 337.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.642.642.64
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z337.920337.920337.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.6731.065-0.000

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Supplemental data

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Sample components

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Entire : AP-1:Arf1:Nef

EntireName: AP-1:Arf1:Nef
Components
  • Sample: AP-1:Arf1:Nef
  • Protein or peptide: adaptor protein-1
  • Protein or peptide: Arf1
  • Protein or peptide: Nef

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Supramolecule #1000: AP-1:Arf1:Nef

SupramoleculeName: AP-1:Arf1:Nef / type: sample / ID: 1000 / Number unique components: 3

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Macromolecule #1: adaptor protein-1

MacromoleculeName: adaptor protein-1 / type: protein_or_peptide / ID: 1 / Name.synonym: AP-1 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightExperimental: 250 KDa / Theoretical: 250 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) pLys / Recombinant plasmid: pST44

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Macromolecule #2: Arf1

MacromoleculeName: Arf1 / type: protein_or_peptide / ID: 2 / Name.synonym: ADP-ribosylation factor-1 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) pLys / Recombinant plasmid: pST44

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Macromolecule #3: Nef

MacromoleculeName: Nef / type: protein_or_peptide / ID: 3 / Name.synonym: negative factor / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightExperimental: 290 KDa / Theoretical: 290 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) pLys / Recombinant plasmid: pST44

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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

Concentration0.2 mg/mL
BufferpH: 8 / Details: 20 mM Tris, 200 mM NaCl, 0.3 mM TCEP
GridDetails: 400 mesh copper grid with thin carbon support
VitrificationCryogen 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

MicroscopeFEI TITAN
DateMar 30, 2015
Image recordingCategory: CCD / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Number real images: 1000 / Average electron dose: 45.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: whole image
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 12000
Final two d classificationNumber classes: 10

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Atomic model buiding 1

Initial modelPDB ID:

4p6z

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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