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- EMDB-5773: A Two-Pronged Structural Analysis of Retroviral Maturation Indica... -

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Entry
Database: EMDB / ID: EMD-5773
TitleA Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition
Map dataReconstruction of RSV-CASP 30 nm outer capsid T=3
Sample
  • Sample: Icosahedral assembly of Rous sarcoma virus capsid proteins with spacer peptide, 30 nm outer particle
  • Virus: Rous sarcoma virus
KeywordsCryo-EM / Rous Sarcoma Virus Structure / in vitro assembled capsids / spacer peptide
Biological speciesRous sarcoma virus
Methodsingle particle reconstruction / cryo EM / Resolution: 18.5 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.
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
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5773.png

emd_5773_1.png

Downloads & links

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Map

FileDownload / File: emd_5773.map.gz / Format: CCP4 / Size: 159.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of RSV-CASP 30 nm outer capsid T=3
Voxel sizeX=Y=Z: 1.27 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-4.2625885 - 6.64531183
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-175-175-175
Dimensions350350350
Spacing350350350
CellA=B=C: 444.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.271.271.27
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z444.500444.500444.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-175-175-175
NC/NR/NS350350350
D min/max/mean-4.2636.6450.000

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Supplemental data

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Sample components

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Entire : Icosahedral assembly of Rous sarcoma virus capsid proteins with s...

EntireName: Icosahedral assembly of Rous sarcoma virus capsid proteins with spacer peptide, 30 nm outer particle
Components
  • Sample: Icosahedral assembly of Rous sarcoma virus capsid proteins with spacer peptide, 30 nm outer particle
  • Virus: Rous sarcoma virus

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Supramolecule #1000: Icosahedral assembly of Rous sarcoma virus capsid proteins with s...

SupramoleculeName: Icosahedral assembly of Rous sarcoma virus capsid proteins with spacer peptide, 30 nm outer particle
type: sample / ID: 1000 / Details: Outer shell of the 30 nm particle / Oligomeric state: icosahedral shell with 120 subunits / Number unique components: 1
Molecular weightTheoretical: 3 MDa

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Supramolecule #1: Rous sarcoma virus

SupramoleculeName: Rous sarcoma virus / type: virus / ID: 1 / NCBI-ID: 11886 / Sci species name: Rous sarcoma virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Gallus gallus (chicken) / synonym: VERTEBRATES
Host systemOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
Virus shellShell ID: 1 / Name: outer capsid / Diameter: 300 Å / T number (triangulation number): 3

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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

Concentration2 mg/mL
BufferpH: 7.5
Details: 10 mM Tris-HCl, 75 mM sodium chloride, 0.05 mM EDTA, 0.5 M sodium phosphate
GridDetails: Holey carbon film on R2/2 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93.15 K / Instrument: LEICA KF80 / Details: Vitrification carried out in nitrogen atmosphere.
Method: 4.0 microliter sample dropped onto grid, blotted on one side for 2 second, then plunged.

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 93.15 K
DateSep 24, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 17 / Average electron dose: 15 e/Å2 / Details: scanning at 4000 dpi / Bits/pixel: 16

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Image processing

CTF correctionDetails: CTF was determined from the whole micrograph. Phase reversal was applied to each particle.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Bsoft / Number images used: 612
Details30 nm particles were picked manually, extracted, and CTF corrected by phase reversal. Particle orientation search and refinement were performed using image package Bsoft.
FSC plot (resolution estimation)

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