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- EMDB-5772: A Two-Pronged Structural Analysis of Retroviral Maturation Indica... -

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Entry
Database: EMDB / ID: EMD-5772
TitleA Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition
Map data
SampleT=1 icosahedral assembly of Rous sarcoma virus capsid proteins with spacer peptide:
virus
KeywordsCryo-EM / Rous Sarcoma Virus Structure / in vitro assembled capsids / spacer peptide
Biological speciesRous sarcoma virus
Methodsingle particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsKeller PW / Huang RK / England M / Waki K / Cheng N / Heymann JB / Craven RC / Freed EO / Steven AC
CitationJournal: J Virol / Year: 2013
Title: A two-pronged structural analysis of retroviral maturation indicates that core formation proceeds by a disassembly-reassembly pathway rather than a displacive transition.
Authors: Paul W Keller / Rick K Huang / Matthew R England / Kayoko Waki / Naiqian Cheng / J Bernard Heymann / Rebecca C Craven / Eric O Freed / Alasdair C Steven /
Abstract: Retrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence ...Retrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence suggests that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persists of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into icosahedral capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionOct 23, 2013-
Header (metadata) releaseNov 6, 2013-
Map releaseNov 6, 2013-
UpdateDec 4, 2013-
Current statusDec 4, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5772.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.27 Å/pix.
x 250 pix.
= 317.5 Å
1.27 Å/pix.
x 250 pix.
= 317.5 Å
1.27 Å/pix.
x 250 pix.
= 317.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.27 Å
Density
Contour LevelBy AUTHOR: 1.1 / Movie #1: 1.1
Minimum - Maximum-2.68567443 - 6.42209005
Average (Standard dev.)0.0 (±0.82537431)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-125-125-125
Dimensions250250250
Spacing250250250
CellA=B=C: 317.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.271.271.27
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z317.500317.500317.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-125-125-125
NC/NR/NS250250250
D min/max/mean-2.6866.422-0.000

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Supplemental data

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Sample components

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Entire T=1 icosahedral assembly of Rous sarcoma virus capsid proteins wi...

EntireName: T=1 icosahedral assembly of Rous sarcoma virus capsid proteins with spacer peptide
Number of components: 1
Oligomeric State: T=1 icosahedral shell with 60 subunits forming 12 pentamers
MassTheoretical: 1.51 MDa

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Component #1: virus, Rous sarcoma virus

VirusName: Rous sarcoma virus / Class: VIRUS-LIKE PARTICLE / Details: icosahedral assembly of CA-SP protein / Empty: Yes / Enveloped: No / Isolate: SPECIES
MassTheoretical: 1.51 MDa
SpeciesSpecies: Rous sarcoma virus
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: BL21
Source (natural)Host Species: Gallus gallus (chicken) / Host category: VERTEBRATES

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/mL
Buffer solution: 10 mM Tris-HCl, 75 mM sodium chloride, 0.05 mM EDTA, 0.5 M sodium phosphate
pH: 7.5
Support filmHoley carbon film on R2/2 400 mesh copper grid
VitrificationInstrument: LEICA KF80 / Cryogen name: ETHANE / Temperature: 93.15 K / Humidity: 90 %
Method: 4.0 microliter sample dropped onto grid, blotted on one side for 2 second, then plunged.
Details: Vitrification carried out in nitrogen atmosphere.

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS CM200FEG / Date: Sep 24, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 15 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 700 - 2000 nm
Specimen HolderModel: GATAN LIQUID NITROGEN / Temperature: 93.15
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 17 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 µm / Bit depth: 16

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 2663
Details: Particles were selected manually and processed with Bsoft.
3D reconstructionAlgorithm: Fourier space projection matching and reconstruction
Software: Bsoft
CTF correction: CTF was determined from the whole micrograph. Phase reversal and baseline correction were applied to each extracted particle.
Resolution: 8.5 Å / Resolution method: FSC 0.333
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Details: RSV CA NTD and CTD structures were rigid-body fitted into the T=1 density map using Chimera fit to map tools.
Input PDB model: 1EM9
Chain ID: A
Modeling #2Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Details: RSV CA NTD and CTD structures were rigid-body fitted into the T=1 density map using Chimera fit to map tools.
Input PDB model: 1EOQ
Chain ID: A

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