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- EMDB-1502: Initial location of the RNA-dependent RNA polymerase in the bacte... -

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Basic information

Entry
Database: EMDB / ID: EMD-1502
TitleInitial location of the RNA-dependent RNA polymerase in the bacteriophage phi6 procapsid determined by cryo-electron microscopy
Map dataMap of a procapsid without the P2 protein, containing only proteins P1, P4 and P7
Sample
  • Sample: Phi 6 procapsid without the P2 protein
  • Virus: Pseudomonas phage phi6 (bacteriophage)
KeywordsCystoviridae / P2 protein
Biological speciesPseudomonas phage phi6 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 11.0 Å
AuthorsSen A / Heymann JB / Cheng N / Qiao J / Mindich L / Steven AC
CitationJournal: J Biol Chem / Year: 2008
Title: Initial location of the RNA-dependent RNA polymerase in the bacteriophage Phi6 procapsid determined by cryo-electron microscopy.
Authors: Anindito Sen / J Bernard Heymann / Naiqian Cheng / Jian Qiao / Leonard Mindich / Alasdair C Steven /
Abstract: The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and ...The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage Phi6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as single-stranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage Phi6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has approximately 10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of approximately 3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.
History
DepositionApr 6, 2008-
Header (metadata) releaseApr 7, 2008-
Map releaseMar 31, 2009-
UpdateNov 7, 2012-
Current statusNov 7, 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
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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

1502.gif

emd_1502.png

emd_1502.tif

emd_1502_1.png

emd_1502_1.tif

Downloads & links

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Map

FileDownload / File: emd_1502.map.gz / Format: CCP4 / Size: 47.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of a procapsid without the P2 protein, containing only proteins P1, P4 and P7
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.54 Å/pix.
x 234 pix.
= 591.82 Å		2.54 Å/pix.
x 234 pix.
= 591.82 Å		2.54 Å/pix.
x 234 pix.
= 591.82 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.54 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 2.25 / Movie #1: 1
Minimum - Maximum-6.38072 - 5.68114
Average (Standard dev.)0.017436 (±0.991402)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-117-117-117
Dimensions234234234
Spacing234234234
CellA=B=C: 591.82 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z233233233
origin x/y/z0.0000.0000.000
length x/y/z591.820591.820591.820
α/β/γ90.00090.00090.000
start NX/NY/NZ494949
NX/NY/NZ969696
MAP C/R/S123
start NC/NR/NS-117-117-117
NC/NR/NS234234234
D min/max/mean-6.3815.6810.017

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Supplemental data

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Sample components

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Entire : Phi 6 procapsid without the P2 protein

EntireName: Phi 6 procapsid without the P2 protein
Components
  • Sample: Phi 6 procapsid without the P2 protein
  • Virus: Pseudomonas phage phi6 (bacteriophage)

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Supramolecule #1000: Phi 6 procapsid without the P2 protein

SupramoleculeName: Phi 6 procapsid without the P2 protein / type: sample / ID: 1000
Details: Produced using the Escherichia coli cell line LM4419 with the plasmid, pLM1906
Oligomeric state: Procapsid with 3 proteins P1, P4, P7 / Number unique components: 1
Molecular weightTheoretical: 14 MDa

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Supramolecule #1: Pseudomonas phage phi6

SupramoleculeName: Pseudomonas phage phi6 / type: virus / ID: 1 / NCBI-ID: 10879 / Sci species name: Pseudomonas phage phi6 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: Yes
Host (natural)Organism: Pseudomonas syringae (bacteria) / synonym: BACTERIA(EUBACTERIA)
Molecular weightTheoretical: 14 MDa
Virus shellShell ID: 1 / Name: Procapsid / Diameter: 470 Å / T number (triangulation number): 1

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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

Concentration0.5 mg/mL
BufferpH: 7.5 / Details: 10mM sodium phosphate, 1mM MgCl2, 150 mM NaCl
StainingType: NEGATIVE / Details: None
VitrificationCryogen name: ETHANE / Instrument: REICHERT-JUNG PLUNGER
Details: Vitrification instrument: Reichert-Jung cryo-fixation unit

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Alignment procedureLegacy - Astigmatism: Corrected at 300000 times magnification
DateMar 15, 2007
Image recordingDigitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 36 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.3 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry, Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Each particle phase flipped
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: FSC 0.33 CUT-OFF / Software - Name: Bsoft, PFT3DR / Number images used: 1072

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