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- EMDB-3571: dsRNA bacteriophage phi6 nucleocapsid -

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Basic information

Entry
Database: EMDB / ID: EMD-3571
TitledsRNA bacteriophage phi6 nucleocapsid
Map datadsRNA bacteriophage phi6 nucleocapsid
Sample
  • Virus: Pseudomonas phage phi6 (bacteriophage)
    • Protein or peptide: Major inner protein P1
    • Protein or peptide: Packaging enzyme P4
    • Protein or peptide: Major outer capsid protein
Function / homology
Function and homology information


T=13 icosahedral viral capsid / T=2 icosahedral viral capsid / viral procapsid / viral genome packaging / viral inner capsid / viral outer capsid / virion component => GO:0044423 / ribonucleoside triphosphate phosphatase activity / viral capsid / nucleoside-triphosphate phosphatase ...T=13 icosahedral viral capsid / T=2 icosahedral viral capsid / viral procapsid / viral genome packaging / viral inner capsid / viral outer capsid / virion component => GO:0044423 / ribonucleoside triphosphate phosphatase activity / viral capsid / nucleoside-triphosphate phosphatase / viral nucleocapsid / RNA binding / ATP binding / identical protein binding
Similarity search - Function
: / Major inner capsid protein P1 / Packaging enzyme P4 / ATPase P4 of dsRNA bacteriophage phi-12 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Major outer capsid protein / Packaging enzyme P4 / Major inner protein P1
Similarity search - Component
Biological speciesPseudomonas phage phi6 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsSun Z / El Omari K / Sun X / Ilca SL / Kotecha A / Stuart DI / Poranen MM / Huiskonen JT
CitationJournal: Nat Commun / Year: 2017
Title: Double-stranded RNA virus outer shell assembly by bona fide domain-swapping.
Authors: Zhaoyang Sun / Kamel El Omari / Xiaoyu Sun / Serban L Ilca / Abhay Kotecha / David I Stuart / Minna M Poranen / Juha T Huiskonen /
Abstract: Correct outer protein shell assembly is a prerequisite for virion infectivity in many multi-shelled dsRNA viruses. In the prototypic dsRNA bacteriophage φ6, the assembly reaction is promoted by ...Correct outer protein shell assembly is a prerequisite for virion infectivity in many multi-shelled dsRNA viruses. In the prototypic dsRNA bacteriophage φ6, the assembly reaction is promoted by calcium ions but its biomechanics remain poorly understood. Here, we describe the near-atomic resolution structure of the φ6 double-shelled particle. The outer T=13 shell protein P8 consists of two alpha-helical domains joined by a linker, which allows the trimer to adopt either a closed or an open conformation. The trimers in an open conformation swap domains with each other. Our observations allow us to propose a mechanistic model for calcium concentration regulated outer shell assembly. Furthermore, the structure provides a prime exemplar of bona fide domain-swapping. This leads us to extend the theory of domain-swapping from the level of monomeric subunits and multimers to closed spherical shells, and to hypothesize a mechanism by which closed protein shells may arise in evolution.
History
DepositionJan 14, 2017-
Header (metadata) releaseJan 25, 2017-
Map releaseMar 22, 2017-
UpdateOct 23, 2019-
Current statusOct 23, 2019Processing 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
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5muu
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5muu
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3571.png

Downloads & links

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Map

FileDownload / File: emd_3571.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdsRNA bacteriophage phi6 nucleocapsid
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.07375267 - 0.14329888
Average (Standard dev.)0.00020935167 (±0.008287506)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 691.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z691.200691.200691.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0740.1430.000

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Supplemental data

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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: Major inner protein P1
    • Protein or peptide: Packaging enzyme P4
    • Protein or peptide: Major outer capsid protein

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Supramolecule #1: Pseudomonas phage phi6

SupramoleculeName: Pseudomonas phage phi6 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The viral envelope was removed by Triton X-114 extraction
NCBI-ID: 10879 / Sci species name: Pseudomonas phage phi6 / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Pseudomonas syringae (bacteria) / Strain: pv.phaseolicola HB10Y
Virus shellShell ID: 1 / Name: Outer shell / Diameter: 565.0 Å / T number (triangulation number): 13
Virus shellShell ID: 2 / Name: Inner shell / Diameter: 500.0 Å / T number (triangulation number): 1

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Macromolecule #1: Major inner protein P1

MacromoleculeName: Major inner protein P1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Molecular weightTheoretical: 85.080711 KDa
SequenceString: MFNLKVKDLN GSARGLTQAF AIGELKNQLS VGALQLPLQF TRTFSASMTS ELLWEVGKGN IDPVMYARLF FQYAQAGGAL SVDELVNQF TEYHQSTACN PEIWRKLTAY ITGSSNRAIK ADAVGKVPPT AILEQLRTLA PSEHELFHHI TTDFVCHVLS P LGFILPDA ...String:
MFNLKVKDLN GSARGLTQAF AIGELKNQLS VGALQLPLQF TRTFSASMTS ELLWEVGKGN IDPVMYARLF FQYAQAGGAL SVDELVNQF TEYHQSTACN PEIWRKLTAY ITGSSNRAIK ADAVGKVPPT AILEQLRTLA PSEHELFHHI TTDFVCHVLS P LGFILPDA AYVYRVGRTA TYPNFYALVD CVRASDLRRM LTALSSVDSK MLQATFKAKG ALAPALISQH LANAATTAFE RS RGNFDAN AVVSSVLTIL GRLWSPSTPK ELDPSARLRN TNGIDQLRSN LALFIAYQDM VKQRGRAEVI FSDEELSSTI IPW FIEAMS EVSPFKLRPI NETTSYIGQT SAIDHMGQPS HVVVYEDWQF AKEITAFTPV KLANNSNQRF LDVEPGISDR MSAT LAPIG NTFAVSAFVK NRTAVYEAVS QRGTVNSNGA EMTLGFPSVV ERDYALDRDP MVAIAALRTG IVDESLEARA SNDLK RSMF NYYAAVMHYA VAHNPEVVVS EHQGVAAEQG SLYLVWNVRT ELRIPVGYNA IEGGSIRTPE PLEAIAYNKP IQPSEV LQA KVLDLANHTT SIHIWPWHEA STEFAYEDAY SVTIRNKRYT AEVKEFELLG LGQRRERVRI LKPTVAHAII QMWYSWF VE DDRTLAAARR TSRDDAEKLA IDGRRMQNAV TLLRKIEMIG TTGIGASAVH LAQSRIVDQM AGRGLIDDSS DLHVGINR H RIRIWAGLAV LQMMGLLSRS EAEALTKVLG DSNALGMVVA TTDIDPSL

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Macromolecule #2: Packaging enzyme P4

MacromoleculeName: Packaging enzyme P4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: nucleoside-triphosphate phosphatase
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Molecular weightTheoretical: 35.198426 KDa
SequenceString: MPIVVTQAHI DRVGIAADLL DASPVSLQVL GRPTAINTVV IKTYIAAVME LASKQGGSLA GVDIRPSVLL KDTAIFTKPK AKSADVESD VDVLDTGIYS VPGLARKPVT HRWPSEGIYS GVTALMGATG SGKSITLNEK LRPDVLIRWG EVAEAYDELD T AVHISTLD ...String:
MPIVVTQAHI DRVGIAADLL DASPVSLQVL GRPTAINTVV IKTYIAAVME LASKQGGSLA GVDIRPSVLL KDTAIFTKPK AKSADVESD VDVLDTGIYS VPGLARKPVT HRWPSEGIYS GVTALMGATG SGKSITLNEK LRPDVLIRWG EVAEAYDELD T AVHISTLD EMLIVCIGLG ALGFNVAVDS VRPLLFRLKG AASAGGIVAV FYSLLTDISN LFTQYDCSVV MVVNPMVDAE KI EYVFGQV MASTVGAILC ADGNVSRTMF RTNKGRIFNG AAPLAADTHM PSMDRPTSMK ALDHTSIASV APLERGSVDT DDR NSAPRR GANFSL

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Macromolecule #3: Major outer capsid protein

MacromoleculeName: Major outer capsid protein / type: protein_or_peptide / ID: 3 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Molecular weightTheoretical: 16.018418 KDa
SequenceString:
MLLPVVARAA VPAIESAIAA TPGLVSRIAA AIGSKVSPSA ILAAVKSNPV VAGLTLAQIG STGYDAYQQL LENHPEVAEM LKDLSFKAD EIQPDFIGNL GQYREELELV EDAARFVGGM SNLIRLRQAL ELDIKYYGLK MQLNDMGYRS

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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

Concentration3 mg/mL
BufferpH: 7.2
GridModel: C-flat / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.0 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.3 µm / Nominal magnification: 160000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 120.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-22 / Number real images: 900 / Average exposure time: 0.2 sec. / Average electron dose: 0.73 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 16466
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 3)CTFFIND was used to determine CTF parameters
RELION (ver. 1.3)RELION was used to apply CTF correction
Startup modelType of model: EMDB MAP
EMDB ID:

1207

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 13291
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4k7h


Chain - Chain ID: A / Chain - Residue range: 1-760
DetailsThe structure of P1 was fitted in the map using COOT as a rigid body in two different positions corresponding to subunits P1A and P1B. P1A and P1B main-chains and side-chains were adjusted using manual and real space fitting in COOT. Structure was refined in Phenix.real_space_refine applying secondary structure, rotamer, and Ramachandran plot restraints.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5muu:
dsRNA bacteriophage phi6 nucleocapsid

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Atomic model buiding 2

DetailsThe structure of P8 (L3 to Y147) was built manually in COOT and side-chains were adjusted using manual and real space fitting in COOT. Structure was refined in Phenix.real_space_refine applying secondary structure, rotamer, and Ramachandran plot restraints.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-5muu:
dsRNA bacteriophage phi6 nucleocapsid

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Atomic model buiding 3

DetailsThe structure of P4 C-terminus (R292 to L332) was built manually in COOT and side-chains were adjusted using manual and real space fitting in COOT. Structure was refined in Phenix.real_space_refine applying secondary structure, rotamer, and Ramachandran plot restraints.
RefinementProtocol: AB INITIO MODEL
Output model

PDB-5muu:
dsRNA bacteriophage phi6 nucleocapsid

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