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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: EMD-0245
TitleStructure of the in vitro assembled bacteriophage phi6 P1P4 complex
Map dataBacteriophage phi6 in vitro assembled P1P4 particle
Sample
  • Virus: Pseudomonas phage phi6 (bacteriophage)
    • Protein or peptide: P1 protein from bacteriophage phi6
    • Protein or peptide: P4 protein from bacteriophage phi6
Biological speciesPseudomonas phage phi6 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
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.
History
DepositionSep 13, 2018-
Header (metadata) releaseSep 26, 2018-
Map releaseOct 17, 2018-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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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

FileDownload / File: emd_0245.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBacteriophage phi6 in vitro assembled P1P4 particle
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.025962658 - 0.07378059
Average (Standard dev.)0.00092696014 (±0.0058279634)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 648.0 Å
α=β=γ: 90.0 °

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
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 (bacteriophage)
Components
  • Virus: Pseudomonas phage phi6 (bacteriophage)
    • Protein or peptide: P1 protein from bacteriophage phi6
    • Protein or peptide: P4 protein from bacteriophage phi6

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Supramolecule #1: Pseudomonas phage phi6

SupramoleculeName: Pseudomonas phage phi6 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 10879 / Sci species name: Pseudomonas phage phi6 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Pseudomonas syringae (bacteria)
Host systemOrganism: Escherichia coli (E. coli) / Recombinant strain: JM109

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Macromolecule #1: P1 protein from bacteriophage phi6

MacromoleculeName: P1 protein from bacteriophage phi6 / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFNLKVKDL NGSARGLTQA FAIGELKNQL SVGALQLPLQ FTRTFSASMT S ELLWEVGK GNIDPVMYAR LFFQYAQAGG ALSVDELVNQ FTEYHQSTAC NP EIWRKLT AYITGSSNRA IKADAVGKVP PTAILEQLRT LAPSEHELFH HIT TDFVCH VLSPLGFILP ...String:
MFNLKVKDL NGSARGLTQA FAIGELKNQL SVGALQLPLQ FTRTFSASMT S ELLWEVGK GNIDPVMYAR LFFQYAQAGG ALSVDELVNQ FTEYHQSTAC NP EIWRKLT AYITGSSNRA IKADAVGKVP PTAILEQLRT LAPSEHELFH HIT TDFVCH VLSPLGFILP DAAYVYRVGR TATYPNFYAL VDCVRASDLR RMLT ALSSV DSKMLQATFK AKGALAPALI SQHLANAATT AFERSRGNFD ANAVV SSVL TILGRLWSPS TPKELDPSAR LRNTNGIDQL RSNLALFIAY QDMVKQ RGR AEVIFSDEEL SSTIIPWFIE AMSEVSPFKL RPINETTSYI GQTSAID HM GQPSHVVVYE DWQFAKEITA FTPVKLANNS NQRFLDVEPG ISDRMSAT L APIGNTFAVS AFVKNRTAVY EAVSQRGTVN SNGAEMTLGF PSVVERDYA LDRDPMVAIA ALRTGIVDES LEARASNDLK RSMFNYYAAV MHYAVAHNPE VVVSEHQGV AAEQGSLYLV WNVRTELRIP VGYNAIEGGS IRTPEPLEAI A YNKPIQPS EVLQAKVLDL ANHTTSIHIW PWHEASTEFA YEDAYSVTIR NK RYTAEVK EFELLGLGQR RERVRILKPT VAHAIIQMWY SWFVEDDRTL AAA RRTSRD DAEKLAIDGR RMQNAVTLLR KIEMIGTTGI GASAVHLAQS RIVD QMAGR GLIDDSSDLH VGINRHRIRI WAGLAVLQMM GLLSRSEAEA LTKVL GDSN ALGMVVATTD IDPSL

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Macromolecule #2: P4 protein from bacteriophage phi6

MacromoleculeName: P4 protein from bacteriophage phi6 / type: protein_or_peptide / ID: 2 / Enantiomer: DEXTRO
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPIVVTQAH IDRVGIAADL LDASPVSLQV LGRPTAINTV VIKTYIAAVM E LASKQGGS LAGVDIRPSV LLKDTAIFTK PKAKSADVES DVDVLDTGIY SV PGLARKP VTHRWPSEGI YSGVTALMGA TGSGKSITLN EKLRPDVLIR WGE VAEAYD ELDTAVHIST ...String:
MPIVVTQAH IDRVGIAADL LDASPVSLQV LGRPTAINTV VIKTYIAAVM E LASKQGGS LAGVDIRPSV LLKDTAIFTK PKAKSADVES DVDVLDTGIY SV PGLARKP VTHRWPSEGI YSGVTALMGA TGSGKSITLN EKLRPDVLIR WGE VAEAYD ELDTAVHIST LDEMLIVCIG LGALGFNVAV DSVRPLLFRL KGAA SAGGI VAVFYSLLTD ISNLFTQYDC SVVMVVNPMV DAEKIEYVFG QVMAS TVGA ILCADGNVSR TMFRTNKGRI FNGAAPLAAD THMPSMDRPT SMKALD HTS IASVAPLERG SVDTDDRNSA PRRGANFSL

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 1.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6878
CTF correctionSoftware - Name: RELION
Startup modelType of model: EMDB MAP
EMDB ID:
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 6857
FSC plot (resolution estimation)

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