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- EMDB-5451: Bacteriophage phi29 prohead particle with a single insertion muta... -

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Basic information

Entry
Database: EMDB / ID: EMD-5451
TitleBacteriophage phi29 prohead particle with a single insertion mutation (U92) in the pRNA
Map dataBacteriophage phi29 prohead particle with a single insertion mutation (U92) in the pRNA
Sample
  • Sample: Bacteriophage phi29 prohead particle with a double insertion mutation (U92,U93) in the pRNA
  • Virus: Bacillus phage phi29 (virus)
Keywordsbacteriophage phi29 / pRNA / DNA packaging
Biological speciesBacillus phage phi29 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.0 Å
AuthorsZhao W / Saha M / Ke A / Morais M / Jardine PJ / Grimes S
CitationJournal: J Virol / Year: 2012
Title: A three-helix junction is the interface between two functional domains of prohead RNA in 29 DNA packaging.
Authors: Wei Zhao / Mitul Saha / Ailong Ke / Marc C Morais / Paul J Jardine / Shelley Grimes /
Abstract: The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage ...The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage 29 has an oligomeric prohead RNA (pRNA) that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we define the functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed that a U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging. Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side of the junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted in a loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA.
History
DepositionJul 24, 2012-
Header (metadata) releaseJul 3, 2013-
Map releaseSep 4, 2013-
UpdateSep 4, 2013-
Current statusSep 4, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5451_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5451.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBacteriophage phi29 prohead particle with a single insertion mutation (U92) in the pRNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.33 Å/pix.
x 300 pix.
= 699. Å		2.33 Å/pix.
x 300 pix.
= 699. Å		2.33 Å/pix.
x 300 pix.
= 699. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.33 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-2.99882269 - 6.22740078
Average (Standard dev.)0.0 (±0.95659858)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions300300300
Spacing300300300
CellA=B=C: 699.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.332.332.33
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z699.000699.000699.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS300300300
D min/max/mean-2.9996.2270.000

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Supplemental data

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Sample components

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Entire : Bacteriophage phi29 prohead particle with a double insertion muta...

EntireName: Bacteriophage phi29 prohead particle with a double insertion mutation (U92,U93) in the pRNA
Components
  • Sample: Bacteriophage phi29 prohead particle with a double insertion mutation (U92,U93) in the pRNA
  • Virus: Bacillus phage phi29 (virus)

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Supramolecule #1000: Bacteriophage phi29 prohead particle with a double insertion muta...

SupramoleculeName: Bacteriophage phi29 prohead particle with a double insertion mutation (U92,U93) in the pRNA
type: sample / ID: 1000
Oligomeric state: The particle is based on a prolate icosahedron with T = 3, Q = 5 quasi-symmetry wherein one pentameric capsomer at one end of the particle is replaced by a dodeameric connector ...Oligomeric state: The particle is based on a prolate icosahedron with T = 3, Q = 5 quasi-symmetry wherein one pentameric capsomer at one end of the particle is replaced by a dodeameric connector protein and a pentameric pRNA
Number unique components: 1
Molecular weightTheoretical: 12.4 MDa
Method: calculated from 255 copies of the capsid protein, 12 copies of the connector protein, and 5 copies of the pRNA

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Supramolecule #1: Bacillus phage phi29

SupramoleculeName: Bacillus phage phi29 / type: virus / ID: 1 / Name.synonym: bacteriophage phi29
Details: The phi29 prohead particle was treated with RNAse to remove the WT pRNA, and then incubated with exogenously produced pRNA engineered to contain a single U92 insertion mutation.
NCBI-ID: 10756 / Sci species name: Bacillus phage phi29 / Database: NCBI / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: Yes / Syn species name: bacteriophage phi29
Host (natural)Organism: Bacillus subtilis (bacteria) / synonym: BACTERIA(EUBACTERIA)
Molecular weightTheoretical: 12.4 MDa

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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

Concentration0.1 mg/mL
BufferpH: 7.8 / Details: 25 mM Tris-HCl, 5 mM MgCl2, 50 mM NaCl
GridDetails: Quantifoil holey carbon on top of 200 mesh copper grid, plasma cleaned
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Method: blot from behind the sample for 4 seconds before plunging

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Electron microscopy

MicroscopeJEOL 2200FS
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Specialist opticsEnergy filter - Name: JEOL
DateNov 24, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 98 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 60000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.54 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe particles were selected using the model-based automatic particle selection implemented in EMAN, followed by manual deletion of bad particles and manual selection of missed particles
CTF correctionDetails: Each Micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN
Details: C5 symmetry was imposed in all stages of the reconstruction, with the 5-fold axis along Z. The final map was low-pass filtered at 14 Angstrom.
Number images used: 1871

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