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- EMDB-1882: Mutually-induced conformational switching of RNA and coat protein... -

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Basic information

Entry
Database: EMDB / ID: EMD-1882
TitleMutually-induced conformational switching of RNA and coat protein underpins efficient assembly of a viral capsid.
Map dataThis is a surface-rendered view of a Bacteriophage MS2 virus-like particle composed of purified coat protein and genomic RNA
Sample
  • Sample: Bacteriophage MS2 virus-like particles assembled in vitro from purified MS2 coat protein and genomic RNA
  • Virus: Enterobacterio phage MS2 (virus)
  • RNA: Single stranded RNA MS2 genome
KeywordsBacteriophage MS2 / virus assembly / quasi-equivalence / ssRNA virus
Biological speciesEnterobacteria phage MS2 (virus) / Enterobacterio phage MS2 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 32.0 Å
AuthorsRolfsson O / Toropova K / Ranson NA / Stockley PG
CitationJournal: J Mol Biol / Year: 2010
Title: Mutually-induced conformational switching of RNA and coat protein underpins efficient assembly of a viral capsid.
Authors: Óttar Rolfsson / Katerina Toropova / Neil A Ranson / Peter G Stockley /
Abstract: Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, ...Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T=3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T=3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.
History
DepositionFeb 21, 2011-
Header (metadata) releaseMay 13, 2011-
Map releaseMay 13, 2011-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
  • Download
  • 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

emd1882.jpg

Downloads & links

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Map

FileDownload / File: emd_1882.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a surface-rendered view of a Bacteriophage MS2 virus-like particle composed of purified coat protein and genomic RNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.87 Å/pix.
x 200 pix.
= 374. Å		1.87 Å/pix.
x 200 pix.
= 374. Å		1.87 Å/pix.
x 200 pix.
= 374. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.87 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 1.0 / Movie #1: 1
Minimum - Maximum-4.45316 - 5.72175
Average (Standard dev.)0.00000000557511 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 374 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.871.871.87
M x/y/z200200200
origin x/y/z-0.000-0.000-0.000
length x/y/z374.000374.000374.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-4.4535.7220.000

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Supplemental data

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Sample components

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Entire : Bacteriophage MS2 virus-like particles assembled in vitro from pu...

EntireName: Bacteriophage MS2 virus-like particles assembled in vitro from purified MS2 coat protein and genomic RNA
Components
  • Sample: Bacteriophage MS2 virus-like particles assembled in vitro from purified MS2 coat protein and genomic RNA
  • Virus: Enterobacterio phage MS2 (virus)
  • RNA: Single stranded RNA MS2 genome

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Supramolecule #1000: Bacteriophage MS2 virus-like particles assembled in vitro from pu...

SupramoleculeName: Bacteriophage MS2 virus-like particles assembled in vitro from purified MS2 coat protein and genomic RNA
type: sample / ID: 1000 / Oligomeric state: One capsid to one RNA / Number unique components: 2

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Supramolecule #1: Enterobacterio phage MS2

SupramoleculeName: Enterobacterio phage MS2 / type: virus / ID: 1 / Name.synonym: MS2 / NCBI-ID: 12022 / Sci species name: Enterobacterio phage MS2 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No / Syn species name: MS2
Host (natural)Organism: Escherichia coli (E. coli) / synonym: BACTERIA(EUBACTERIA)
Virus shellShell ID: 1 / Diameter: 280 Å / T number (triangulation number): 3

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Macromolecule #1: Single stranded RNA MS2 genome

MacromoleculeName: Single stranded RNA MS2 genome / type: rna / ID: 1 / Name.synonym: Single stranded RNA MS2 genome
Details: Single stranded RNA MS2 genome was phenol-chloroform extracted from wild type MS2 phage
Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Enterobacteria phage MS2 (virus) / synonym: MS2

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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

BufferpH: 7.6
Details: 50mM Tris-acetate, 1mM magnesium acetate, 40mM ammonium acetate
GridDetails: Quantifoil R2/1 200 mesh holly carbon grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Double-sided custom pneumatic blotter
Method: 1.6s blot

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 9.88 µm / Average electron dose: 18 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 52750 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flip each particle
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 80
Final two d classificationNumber classes: 19

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