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- EMDB-2364: CryoEM reconstruction of the bacteriophage phi6 procapsid to the ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2364
TitleCryoEM reconstruction of the bacteriophage phi6 procapsid to the near-atomic resolution
Map dataReconstruction of the wildtype P1247 procapsid of bacteriophage phi6
Sample
  • Sample: Wildtype P1247 procapsid of bacteriophage phi6
  • Virus: Pseudomonas phage phi6 (bacteriophage)
KeywordsBacteriophage phi6 / Cystoviridae / capsid structure / capsid expansion / segmented genome / conformational change / RNA packaging
Function / homology: / Major inner capsid protein P1 / T=2 icosahedral viral capsid / viral inner capsid / viral nucleocapsid / RNA binding / identical protein binding / Major inner protein P1
Function and homology information
Biological speciesPseudomonas phage phi6 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsNemecek D / Boura E / Wu W / Cheng N / Plevka P / Qiao J / Mindich L / Heymann JB / Hurley JH / Steven AC
CitationJournal: Structure / Year: 2013
Title: Subunit folds and maturation pathway of a dsRNA virus capsid.
Authors: Daniel Nemecek / Evzen Boura / Weimin Wu / Naiqian Cheng / Pavel Plevka / Jian Qiao / Leonard Mindich / J Bernard Heymann / James H Hurley / Alasdair C Steven /
Abstract: The cystovirus ϕ6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits ...The cystovirus ϕ6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. ϕ6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an α-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.
History
DepositionApr 17, 2013-
Header (metadata) releaseApr 24, 2013-
Map releaseAug 14, 2013-
UpdateAug 21, 2013-
Current statusAug 21, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4btg
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4btg
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2364.jpg

Downloads & links

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Map

FileDownload / File: emd_2364.map.gz / Format: CCP4 / Size: 276 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the wildtype P1247 procapsid of bacteriophage phi6
Voxel sizeX=Y=Z: 1.397 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-8.04054451 - 21.12542534
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-210-210-210
Dimensions420420420
Spacing420420420
CellA=B=C: 586.74 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3971.3971.397
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z586.740586.740586.740
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-210-210-210
NC/NR/NS420420420
D min/max/mean-8.04121.125-0.000

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Supplemental data

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Segmentation: This mask represents the P1B subunit

AnnotationThis mask represents the P1B subunit
Fileemd_2364_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: This mask represents the P1A subunit

AnnotationThis mask represents the P1A subunit
Fileemd_2364_msk_2.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 : Wildtype P1247 procapsid of bacteriophage phi6

EntireName: Wildtype P1247 procapsid of bacteriophage phi6
Components
  • Sample: Wildtype P1247 procapsid of bacteriophage phi6
  • Virus: Pseudomonas phage phi6 (bacteriophage)

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Supramolecule #1000: Wildtype P1247 procapsid of bacteriophage phi6

SupramoleculeName: Wildtype P1247 procapsid of bacteriophage phi6 / type: sample / ID: 1000 / Oligomeric state: icosahedral shell with accessory proteins / Number unique components: 4
Molecular weightTheoretical: 12.6 MDa

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Supramolecule #1: Pseudomonas phage phi6

SupramoleculeName: Pseudomonas phage phi6 / type: virus / ID: 1 / Name.synonym: bacteriophage phi-6 / NCBI-ID: 10879 / Sci species name: Pseudomonas phage phi6 / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: Yes / Syn species name: bacteriophage phi-6
Host (natural)Organism: Pseudomonas syringae (bacteria) / synonym: BACTERIA(EUBACTERIA)
Host systemOrganism: Escherichia coli (E. coli) / Recombinant strain: JM109 / Recombinant plasmid: pLM687
Molecular weightTheoretical: 12.6 MDa
Virus shellShell ID: 1 / Name: P1247 / Diameter: 450 Å / T number (triangulation number): 2

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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

Concentration10 mg/mL
BufferpH: 8 / Details: 10 mM potassium phosphate, 5 mM MgCl2
GridDetails: 400 mesh C-flat holey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK I / Method: Blot for 2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 44739 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 46000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateNov 20, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 220 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Particles from each micrograph
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, BSOFT / Number images used: 18326
DetailsThe particles were selected using e2boxer (EMAN) and manually pruned in bshow (BSOFT). The initial model was taken from our previous reconstruction at 7A resolution (EMD-2341). The final structure was reconstructed in EMAN.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4k7h


Chain - Chain ID: A
SoftwareName: Chimera, MDFF, Coot
Detailsthe P1 crystal structure was rigid body-fitted into the procapsid map using Chimera. Regions where the crystal structure deviated significantly from the EM density were roughly adjusted in Coot and then the P1A and P1B structures were flexibly fitted using the MDFF package. Finally, both structures were visually inspected and refined in Coot.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4btg:
Coordinates of the bacteriophage phi6 capsid subunits (P1A and P1B) fitted into the cryoEM reconstruction of the procapsid at 4.4 A resolution

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