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- EMDB-3185: Structure of the in vitro assembled bacteriophage phi6 polymerase... -

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Basic information

Entry
Database: EMDB / ID: EMD-3185
TitleStructure of the in vitro assembled bacteriophage phi6 polymerase complex
Map dataIcosahedral reconstruction of in vitro assembled bacteriophage phi6 polymerase complex
Sample
  • Sample: Bacteriophage phi6 polymerase complex assembled in vitro from purified proteins P1, P2, and P4
  • Protein or peptide: P1 protein from bacteriophage phi6
  • Protein or peptide: P2 protein from bacteriophage phi6
  • Protein or peptide: P4 protein from bacteriophage phi6
KeywordsBacteriophage phi6 / polymerase complex
Function / homology
Function and homology information


T=2 icosahedral viral capsid / viral procapsid / RNA uridylyltransferase activity / viral genome packaging / viral inner capsid / ribonucleoside triphosphate phosphatase activity / virion component / nucleoside-triphosphate phosphatase / viral capsid / viral nucleocapsid ...T=2 icosahedral viral capsid / viral procapsid / RNA uridylyltransferase activity / viral genome packaging / viral inner capsid / ribonucleoside triphosphate phosphatase activity / virion component / nucleoside-triphosphate phosphatase / viral capsid / viral nucleocapsid / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding / ATP binding / identical protein binding / metal ion binding
Similarity search - Function
: / Major inner capsid protein P1 / Cystovirus, RNA-directed RNA polymerase, N-terminal / Cystovirus, RNA-directed RNA polymerase / Packaging enzyme P4 / ATPase P4 of dsRNA bacteriophage phi-12 / RNA-directed RNA polymerase, bacteriophage, catalytic domain / RdRp of RNA-containing bacteriophages catalytic domain profile. / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase ...: / Major inner capsid protein P1 / Cystovirus, RNA-directed RNA polymerase, N-terminal / Cystovirus, RNA-directed RNA polymerase / Packaging enzyme P4 / ATPase P4 of dsRNA bacteriophage phi-12 / RNA-directed RNA polymerase, bacteriophage, catalytic domain / RdRp of RNA-containing bacteriophages catalytic domain profile. / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA-directed RNA polymerase / Packaging enzyme P4 / Major inner protein P1
Similarity search - Component
Biological speciesPseudomonas phage phi6 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsIlca S / Kotecha A / Sun X / Poranen MP / Stuart DI / Huiskonen JT
CitationJournal: Nat Commun / Year: 2015
Title: Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes.
Authors: Serban L Ilca / Abhay Kotecha / Xiaoyu Sun / Minna M Poranen / David I Stuart / Juha T Huiskonen /
Abstract: Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the ...Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.
History
DepositionOct 3, 2015-
Header (metadata) releaseOct 28, 2015-
Map releaseNov 4, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing 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-5fj5
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5fj5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3185.tiff

emd_3185.tiff

Downloads & links

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Map

FileDownload / File: emd_3185.map.gz / Format: CCP4 / Size: 412 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIcosahedral reconstruction of in vitro assembled bacteriophage phi6 polymerase complex
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.04862845 - 0.05562008
Average (Standard dev.)-0.0000143 (±0.00450121)
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-0.0490.056-0.000

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Supplemental data

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Segmentation: Mask used for FSC

AnnotationMask used for FSC
Fileemd_3185_msk_1.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 : Bacteriophage phi6 polymerase complex assembled in vitro from pur...

EntireName: Bacteriophage phi6 polymerase complex assembled in vitro from purified proteins P1, P2, and P4
Components
  • Sample: Bacteriophage phi6 polymerase complex assembled in vitro from purified proteins P1, P2, and P4
  • Protein or peptide: P1 protein from bacteriophage phi6
  • Protein or peptide: P2 protein from bacteriophage phi6
  • Protein or peptide: P4 protein from bacteriophage phi6

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Supramolecule #1000: Bacteriophage phi6 polymerase complex assembled in vitro from pur...

SupramoleculeName: Bacteriophage phi6 polymerase complex assembled in vitro from purified proteins P1, P2, and P4
type: sample / ID: 1000
Oligomeric state: Icosahedral assembly with 120 copies of P1
Number unique components: 3

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Macromolecule #1: P1 protein from bacteriophage phi6

MacromoleculeName: P1 protein from bacteriophage phi6 / type: protein_or_peptide / ID: 1 / Number of copies: 120
Oligomeric state: 60 asymmetric dimers from an icosahedral shell
Recombinant expression: Yes
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Molecular weightTheoretical: 85 KDa
Recombinant expressionOrganism: Pseudomonas syringae (bacteria) / Recombinant strain: pathovar phaseolicola / Recombinant plasmid: pLM358
SequenceUniProtKB: Major inner protein P1

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Macromolecule #2: P2 protein from bacteriophage phi6

MacromoleculeName: P2 protein from bacteriophage phi6 / type: protein_or_peptide / ID: 2 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage) / synonym: bacteriophage phi6
Molecular weightTheoretical: 75 KDa
Recombinant expressionOrganism: Pseudomonas syringae (bacteria) / Recombinant strain: pathovar phaseolicola / Recombinant plasmid: pLM358
SequenceUniProtKB: RNA-directed RNA polymerase

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Macromolecule #3: P4 protein from bacteriophage phi6

MacromoleculeName: P4 protein from bacteriophage phi6 / type: protein_or_peptide / ID: 3 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage) / synonym: bacteriophage phi6
Molecular weightTheoretical: 35 KDa
Recombinant expressionOrganism: Pseudomonas syringae (bacteria) / Recombinant strain: pathovar phaseolicola / Recombinant plasmid: pLM358
SequenceUniProtKB: Packaging enzyme P4

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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

Concentration2.4 mg/mL
BufferpH: 8 / Details: 50 mM Tris
GridDetails: glow discharged Cflat grid (CF-2/1-2C-T)
VitrificationCryogen name: ETHANE / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: Blot 4 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 81 K / Max: 120 K / Average: 81 K
Specialist opticsEnergy filter - Name: GIF QUANTUM LS / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Detailsdose rate 6-8 e-/pix/s
DateJun 12, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 834 / Average electron dose: 16 e/Å2
Details: Every image is the average of 22 frames recorded by the direct electron detector
Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 160000
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsParticles were selected from the best classes after 2D and 3D classification
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: OTHER / Software - Name: Relion
Details: Final map was postprocessed in Relion. Inverse B-factor of 200 was applied.
Number images used: 4379
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4k7h


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera, COOT, Phenix
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5fj5:
Structure of the in vitro assembled bacteriophage phi6 polymerase complex

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