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- EMDB-8306: CryoEM structure of a beak and feather disease virus-like particl... -

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Basic information

Entry
Database: EMDB / ID: EMD-8306
TitleCryoEM structure of a beak and feather disease virus-like particle encapsidating ssDNA
Map dataBeak and feather disease virus-like particle containing ssDNA
Sample
  • Virus: Beak and feather disease virus
    • Organelle or cellular component: 42mer ssDNA
      • DNA: 42mer ssDNA
    • Complex: Cap protein
      • Protein or peptide: Cap Protein
Biological speciesBeak and feather disease virus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsHardy JM / Sarker S / Radjainia M / Raidal SR / Forwood JK / Coulibaly F
CitationJournal: Nat Commun / Year: 2016
Title: Structural insights into the assembly and regulation of distinct viral capsid complexes.
Authors: Subir Sarker / María C Terrón / Yogesh Khandokar / David Aragão / Joshua M Hardy / Mazdak Radjainia / Manuel Jiménez-Zaragoza / Pedro J de Pablo / Fasséli Coulibaly / Daniel Luque / ...Authors: Subir Sarker / María C Terrón / Yogesh Khandokar / David Aragão / Joshua M Hardy / Mazdak Radjainia / Manuel Jiménez-Zaragoza / Pedro J de Pablo / Fasséli Coulibaly / Daniel Luque / Shane R Raidal / Jade K Forwood /
Abstract: The assembly and regulation of viral capsid proteins into highly ordered macromolecular complexes is essential for viral replication. Here, we utilize crystal structures of the capsid protein from ...The assembly and regulation of viral capsid proteins into highly ordered macromolecular complexes is essential for viral replication. Here, we utilize crystal structures of the capsid protein from the smallest and simplest known viruses capable of autonomously replicating in animal cells, circoviruses, to establish structural and mechanistic insights into capsid morphogenesis and regulation. The beak and feather disease virus, like many circoviruses, encode only two genes: a capsid protein and a replication initiation protein. The capsid protein forms distinct macromolecular assemblies during replication and here we elucidate these structures at high resolution, showing that these complexes reverse the exposure of the N-terminal arginine rich domain responsible for DNA binding and nuclear localization. We show that assembly of these complexes is regulated by single-stranded DNA (ssDNA), and provide a structural basis of capsid assembly around single-stranded DNA, highlighting novel binding interfaces distinct from the highly positively charged N-terminal ARM domain.
History
DepositionAug 2, 2016-
Header (metadata) releaseOct 5, 2016-
Map releaseOct 12, 2016-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8306.png

Downloads & links

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Map

FileDownload / File: emd_8306.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBeak and feather disease virus-like particle containing ssDNA
Voxel sizeX=Y=Z: 1.02 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.022331327 - 0.08072096
Average (Standard dev.)0.0032806823 (±0.01173152)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 265.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.021.021.02
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z265.200265.200265.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.0220.0810.003

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Supplemental data

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Additional map: The difference map produced by subtracting the crystal...

Fileemd_8306_additional.map
AnnotationThe difference map produced by subtracting the crystal structure of the protein component from the cryoEM map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Beak and feather disease virus

EntireName: Beak and feather disease virus
Components
  • Virus: Beak and feather disease virus
    • Organelle or cellular component: 42mer ssDNA
      • DNA: 42mer ssDNA
    • Complex: Cap protein
      • Protein or peptide: Cap Protein

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Supramolecule #1: Beak and feather disease virus

SupramoleculeName: Beak and feather disease virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Recombinant protein complex bound to ssDNA in an icosahedral arrangement
NCBI-ID: 77856 / Sci species name: Beak and feather disease virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Cacatua leadbeateri (Major Mitchell's cockatoo)
Host systemOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2 / Recombinant plasmid: pMCSG21
Molecular weightTheoretical: 2.595 MDa
Virus shellShell ID: 1 / Name: Capsid / Diameter: 170.0 Å / T number (triangulation number): 1

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Supramolecule #2: 42mer ssDNA

SupramoleculeName: 42mer ssDNA / type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #2
Details: The difference map produced by subtracting the crystal structure of the protein component (PDB: 5J37) from the cryoEM map
Source (natural)Organism: Beak and feather disease virus
Molecular weightTheoretical: 784 KDa

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Supramolecule #3: Cap protein

SupramoleculeName: Cap protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1 / Details: Icosahedral capsid
Source (natural)Organism: Beak and feather disease virus
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2 / Recombinant plasmid: pMCSG21
Molecular weightTheoretical: 1.811 MDa

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Macromolecule #1: Cap Protein

MacromoleculeName: Cap Protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Beak and feather disease virus
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHSSG VDLGTENLYF QSNARRRYAR PYRRRHIRRY RRRRRHFRRR RFSTNRIYTL RLTRQFQFKI NKQTTSVGNL IFNADYITFA LDDFLQAVPN PHTLNFEDYR IKLAKMEMRP TGGHYTVQSD GFGHTAVIQD SRITRFKTTA DQTQDPLAPF DGAKKWFVSR ...String:
MHHHHHHSSG VDLGTENLYF QSNARRRYAR PYRRRHIRRY RRRRRHFRRR RFSTNRIYTL RLTRQFQFKI NKQTTSVGNL IFNADYITFA LDDFLQAVPN PHTLNFEDYR IKLAKMEMRP TGGHYTVQSD GFGHTAVIQD SRITRFKTTA DQTQDPLAPF DGAKKWFVSR GFKRLLRPKP QITIEDLTTA NQSAALWLNS ARTGWIPLQG GPNSAGTKVR HYGIAFSFPQ PEQTITYVTK LTLYVQFRQF APNNPST

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Macromolecule #2: 42mer ssDNA

MacromoleculeName: 42mer ssDNA / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: Beak and feather disease virus
SequenceString:
TTATCCACTT CCAATGTTAT TAAGTGCTGG GATTGTTAGG GG

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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

Concentration3.6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
125.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 3 second blot (-1 force), 2 second drain time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 127000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Sampling interval: 14.0 µm / Digitization - Frames/image: 1-6 / Number grids imaged: 1 / Number real images: 1153 / Average exposure time: 1.0 sec. / Average electron dose: 2.65 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 64278
CTF correctionSoftware: (Name: CTFFIND (ver. 3), RELION (ver. 1.4))
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5j37

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 22663
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsFitting, considering the icosahedral symmetries, was done using UCSF Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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