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- EMDB-2356: Negative staining three-dimensional reconstruction of bacteriopha... -

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Basic information

Entry
Database: EMDB / ID: EMD-2356
TitleNegative staining three-dimensional reconstruction of bacteriophage T7 connector/terminase complex
Map dataReconstruction of the connector/terminase complex, which presents two different symmetries (c12 and c5 respectively).
Sample
  • Sample: Connector/terminase complex of bacteriophage T7
  • Protein or peptide: connector
  • Protein or peptide: large terminase
Keywordspackaging motor / connector / terminase / bacteriophage / DNA translocation / Packaging / single-particle reconstruction / ATPase.
Function / homology
Function and homology information


: / virion component => GO:0044423 / viral terminase, large subunit / viral portal complex / symbiont genome ejection through host cell envelope, short tail mechanism / viral DNA genome packaging / nuclease activity / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / chromosome organization / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement ...: / virion component => GO:0044423 / viral terminase, large subunit / viral portal complex / symbiont genome ejection through host cell envelope, short tail mechanism / viral DNA genome packaging / nuclease activity / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / chromosome organization / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / endonuclease activity / nucleotide binding / ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
Terminase, large subunit gp19 / : / Terminase Bacteriophage T7, Ribonuclease H-like domain / Portal protein, Caudovirales / Head-to-tail connector protein, podovirus-type / Bacteriophage head to tail connecting protein / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Terminase, large subunit / Portal protein
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus)
Methodsingle particle reconstruction / negative staining / Resolution: 29.0 Å
AuthorsDauden MI / Martin-Benito J / Sanchez-Ferrero JC / Pulido-Cid M / Valpuesta JM / Carrascosa JL
CitationJournal: J Biol Chem / Year: 2013
Title: Large terminase conformational change induced by connector binding in bacteriophage T7.
Authors: María I Daudén / Jaime Martín-Benito / Juan C Sánchez-Ferrero / Mar Pulido-Cid / José M Valpuesta / José L Carrascosa /
Abstract: During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead ...During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.
History
DepositionApr 10, 2013-
Header (metadata) releaseApr 24, 2013-
Map releaseMay 8, 2013-
UpdateJun 19, 2013-
Current statusJun 19, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0248
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0248
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4bil
  • Surface level: 0.0248
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4bil
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2356.png

Downloads & links

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Map

FileDownload / File: emd_2356.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the connector/terminase complex, which presents two different symmetries (c12 and c5 respectively).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.2 Å/pix.
x 112 pix.
= 470.4 Å		4.2 Å/pix.
x 112 pix.
= 470.4 Å		4.2 Å/pix.
x 112 pix.
= 470.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0248 / Movie #1: 0.0248
Minimum - Maximum-0.02651265 - 0.06766804
Average (Standard dev.)-0.00067221 (±0.00622457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions112112112
Spacing112112112
CellA=B=C: 470.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z470.400470.400470.400
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS112112112
D min/max/mean-0.0270.068-0.001

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Supplemental data

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Sample components

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Entire : Connector/terminase complex of bacteriophage T7

EntireName: Connector/terminase complex of bacteriophage T7
Components
  • Sample: Connector/terminase complex of bacteriophage T7
  • Protein or peptide: connector
  • Protein or peptide: large terminase

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Supramolecule #1000: Connector/terminase complex of bacteriophage T7

SupramoleculeName: Connector/terminase complex of bacteriophage T7 / type: sample / ID: 1000
Oligomeric state: one homododecamer bound to one homopentamer
Number unique components: 2
Molecular weightTheoretical: 1.0 MDa

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Macromolecule #1: connector

MacromoleculeName: connector / type: protein_or_peptide / ID: 1 / Name.synonym: portal protein, gp8 / Details: Each monomer of the connector is a 59 kDa protein. / Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: pLys / Recombinant plasmid: pET28Aa
SequenceUniProtKB: Portal protein / GO: virion component => GO:0044423

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Macromolecule #2: large terminase

MacromoleculeName: large terminase / type: protein_or_peptide / ID: 2 / Name.synonym: gp19 / Details: Each monomer of the terminase is a 67 kDa protein / Number of copies: 5 / Oligomeric state: Pentamer / Recombinant expression: Yes
Source (natural)Organism: Enterobacteria phage T7 (virus) / synonym: bacteriophage T7
Molecular weightTheoretical: 335 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: pLys / Recombinant plasmid: pET21(+)
SequenceUniProtKB: Terminase, large subunit
GO: GO: 0090305, nucleotide binding, nuclease activity, endonuclease activity, ATP binding

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: 50 mM Sodium Phosphate buffer pH 7, 300 mM NaCl, 10 mM MgCl2, 1 mM ADP, 5 mM DTT and 20% (v/v) glycerol.
StainingType: NEGATIVE
Details: GraFix-fixated proteins stained on 2% w/v uranyl acetate for 1 min.
GridDetails: Cu-collodion grids with thin carbon support, glow discharged.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: FFT live
DateJul 7, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 573 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 67000
Sample stageSpecimen holder: GATAN conventional holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using Maximum Likelihood procedures form an initial set of 10034 particles.
CTF correctionDetails: Each plate
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 29.0 Å / Resolution method: FSC 0.33 CUT-OFF / Software - Name: XMIPP, EMAN, Spider / Number images used: 837
Final angle assignmentDetails: SPIDER

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Atomic model buiding 1

Initial modelPDB ID:

3cpe


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E
SoftwareName: Chimera, SITUS
Details3CPE PDB was used as a model for the generation of the threading model of gp19 in the complex conformation.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4bil:
Threading model of the T7 large terminase within the gp8gp19 complex

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