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- EMDB-0245: Structure of the in vitro assembled bacteriophage phi6 P1P4 complex -

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Basic information

Entry
Database: EMDB / ID: 0245
TitleStructure of the in vitro assembled bacteriophage phi6 P1P4 complex
Map dataBacteriophage phi6 in vitro assembled P1P4 particle
SamplePseudomonas phage phi6Cystovirus:
virus / P1 protein from bacteriophage phi6 / P4 protein from bacteriophage phi6
SourcePseudomonas phage phi6 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / 4.8 Å resolution
AuthorsHuiskonen JT / Ilca SL
CitationJournal: MBio / Year: 2018
Title: Dual Role of a Viral Polymerase in Viral Genome Replication and Particle Self-Assembly.
Authors: Xiaoyu Sun / Serban L Ilca / Juha T Huiskonen / Minna M Poranen
Abstract: Double-stranded RNA (dsRNA) viruses package several RNA-dependent RNA polymerases (RdRp) together with their dsRNA genome into an icosahedral protein capsid known as the polymerase complex. This ...Double-stranded RNA (dsRNA) viruses package several RNA-dependent RNA polymerases (RdRp) together with their dsRNA genome into an icosahedral protein capsid known as the polymerase complex. This structure is highly conserved among dsRNA viruses but is not found in any other virus group. RdRp subunits typically interact directly with the main capsid proteins, close to the 5-fold symmetric axes, and perform viral genome replication and transcription within the icosahedral protein shell. In this study, we utilized phage Φ6, a well-established virus self-assembly model, to probe the potential roles of the RdRp in dsRNA virus assembly. We demonstrated that Φ6 RdRp accelerates the polymerase complex self-assembly process and contributes to its conformational stability and integrity. We highlight the role of specific amino acid residues on the surface of the RdRp in its incorporation during the self-assembly reaction. Substitutions of these residues reduce RdRp incorporation into the polymerase complex during the self-assembly reaction. Furthermore, we determined that the overall transcription efficiency of the Φ6 polymerase complex increased when the number of RdRp subunits exceeded the number of genome segments. These results suggest a mechanism for RdRp recruitment in the polymerase complex and highlight its novel role in virion assembly, in addition to the canonical RNA transcription and replication functions. Double-stranded RNA viruses infect a wide spectrum of hosts, including animals, plants, fungi, and bacteria. Yet genome replication mechanisms of these viruses are conserved. During the infection cycle, a proteinaceous capsid, the polymerase complex, is formed. An essential component of this capsid is the viral RNA polymerase that replicates and transcribes the enclosed viral genome. The polymerase complex structure is well characterized for many double-stranded RNA viruses. However, much less is known about the hierarchical molecular interactions that take place in building up such complexes. Using the bacteriophage Φ6 self-assembly system, we obtained novel insights into the processes that mediate polymerase subunit incorporation into the polymerase complex for generation of functional structures. The results presented pave the way for the exploitation and engineering of viral self-assembly processes for biomedical and synthetic biology applications. An understanding of viral assembly processes at the molecular level may also facilitate the development of antivirals that target viral capsid assembly.
DateDeposition: Sep 13, 2018 / Header (metadata) release: Sep 26, 2018 / Map release: Oct 17, 2018 / Last update: Oct 17, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0245.map.gz (map file in CCP4 format, 442369 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
480 pix
1.35 Å/pix.
= 648. Å
480 pix
1.35 Å/pix.
= 648. Å
480 pix
1.35 Å/pix.
= 648. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density
Contour Level:0.02 (by author), 0.02 (movie #1):
Minimum - Maximum-0.025962658 - 0.07378059
Average (Standard dev.)0.00092696014 (0.0058279634)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions480480480
Origin0.00.00.0
Limit479.0479.0479.0
Spacing480480480
CellA=B=C: 648.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z648.000648.000648.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.0260.0740.001

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Supplemental data

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Mask #1

Fileemd_0245_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Pseudomonas phage phi6

EntireName: Pseudomonas phage phi6 / Number of components: 3

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Component #1: virus, Pseudomonas phage phi6

VirusName: Pseudomonas phage phi6Cystovirus / Class: VIRUS-LIKE PARTICLE / Empty: Yes / Enveloped: No / Isolate: OTHER
SpeciesSpecies: Pseudomonas phage phi6 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: JM109
Source (natural)Host Species: Pseudomonas syringae (bacteria)

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Component #2: protein, P1 protein from bacteriophage phi6

ProteinName: P1 protein from bacteriophage phi6 / Recombinant expression: No
SourceSpecies: Pseudomonas phage phi6 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, P4 protein from bacteriophage phi6

ProteinName: P4 protein from bacteriophage phi6 / Recombinant expression: No
SourceSpecies: Pseudomonas phage phi6 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum LS
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 6857
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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