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- EMDB-5738: Electron cryo-microscopy of 2H15BMV -

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Basic information

Entry
Database: EMDB / ID: EMD-5738
TitleElectron cryo-microscopy of 2H15BMV
Map datareconstruction of BMV 2H mutant
Sample
  • Sample: 2H15 BMV
  • Virus: Brome mosaic virus
KeywordsBrome Mosaic Virus / protein / capsid / RNA encapsidation / electrostatic interaction
Biological speciesBrome mosaic virus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 5.0 Å
AuthorsNi P / Wang Z / Ma X / Das NC / Sokol P / Chiu W / Dragnea B / Hagan M / Kao CC
CitationJournal: J Mol Biol / Year: 2012
Title: An examination of the electrostatic interactions between the N-terminal tail of the Brome Mosaic Virus coat protein and encapsidated RNAs.
Authors: Peng Ni / Zhao Wang / Xiang Ma / Nayaran Chandra Das / Paul Sokol / Wah Chiu / Bogdan Dragnea / Michael Hagan / C Cheng Kao /
Abstract: The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction ...The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ∼900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.
History
DepositionAug 7, 2013-
Header (metadata) releaseMar 19, 2014-
Map releaseMar 19, 2014-
UpdateMar 19, 2014-
Current statusMar 19, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5738.gif

Downloads & links

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Map

FileDownload / File: emd_5738.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of BMV 2H mutant
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 256 pix.
= 332.8 Å		1.3 Å/pix.
x 256 pix.
= 332.8 Å		1.3 Å/pix.
x 256 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.01 / Movie #1: 0.012
Minimum - Maximum-0.04041042 - 0.04116914
Average (Standard dev.)0.0 (±0.00562874)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-98-98-98
Dimensions256256256
Spacing256256256
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-98-98-98
NC/NR/NS256256256
D min/max/mean-0.0400.041-0.000

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Supplemental data

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Sample components

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Entire : 2H15 BMV

EntireName: 2H15 BMV
Components
  • Sample: 2H15 BMV
  • Virus: Brome mosaic virus

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Supramolecule #1000: 2H15 BMV

SupramoleculeName: 2H15 BMV / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 4.6 MDa

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Supramolecule #1: Brome mosaic virus

SupramoleculeName: Brome mosaic virus / type: virus / ID: 1 / NCBI-ID: 12302 / Sci species name: Brome mosaic virus / Database: NCBI / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Nicotiana benthamiana (plant) / synonym: PLANTAE(HIGHER PLANTS)
Molecular weightExperimental: 4.6 MDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 5.2
Details: virus 928 buffer II (50 mM NaOAc, 10 mM MgCl2, pH 5.2), 50% w/v CsCl
StainingType: NEGATIVE / Details: 1.2/1.3 Quantifoil grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 20 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2 seconds.

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Electron microscopy

MicroscopeJEOL 3200FSC
DateSep 11, 2009
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 471
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal magnification: 80000
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER

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Image processing

DetailsParticles were selected from electron micrographs using the EMAN Boxer routine. The particles were translationally and rotationally aligned, classified, and averaged without applying symmetry using the EMAN Refine2d command. Initial model for reconstruction: approximately 3000 particles were selected by MPSA (Multi-Path Simulated Annealing) to build a subnanometer model of 2H15. The map was then refined using EMAN.
CTF correctionDetails: each image
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: mpsa, EMAN1.8 / Number images used: 11576

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Atomic model buiding 1

Initial modelPDB ID:

1js9

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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