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- EMDB-2355: Negative staining three dimensional reconstruction of bacteriopha... -

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Basic information

Entry
Database: EMDB / ID: EMD-2355
TitleNegative staining three dimensional reconstruction of bacteriophage T7 large terminase
Map dataThreedimensional reconstruction of bacteriophage T7 large terminase
Sample
  • Sample: Full-length large terminase of bacteriophage T7
  • Protein or peptide: DNA maturase B
KeywordsATPase / bacteriophage / DNA translocation / Packaging / single-particle reconstruction / terminase.
Function / homology
Function and homology information


viral terminase, large subunit / 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 ...viral terminase, large subunit / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Terminase, large subunit
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus)
Methodsingle particle reconstruction / negative staining / Resolution: 16.0 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: 0.0383
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0383
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4bij
  • Surface level: 0.0383
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2355.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThreedimensional reconstruction of bacteriophage T7 large terminase
Voxel sizeX=Y=Z: 4.4 Å
Density
Contour LevelBy AUTHOR: 0.0383 / Movie #1: 0.0383
Minimum - Maximum-0.05090131 - 0.09429953
Average (Standard dev.)0.00008561 (±0.00896379)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.44.44.4
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z352.000352.000352.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0510.0940.000

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Supplemental data

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Sample components

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Entire : Full-length large terminase of bacteriophage T7

EntireName: Full-length large terminase of bacteriophage T7
Components
  • Sample: Full-length large terminase of bacteriophage T7
  • Protein or peptide: DNA maturase B

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Supramolecule #1000: Full-length large terminase of bacteriophage T7

SupramoleculeName: Full-length large terminase of bacteriophage T7 / type: sample / ID: 1000 / Oligomeric state: pentamer / Number unique components: 1
Molecular weightTheoretical: 335 KDa

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Macromolecule #1: DNA maturase B

MacromoleculeName: DNA maturase B / type: protein_or_peptide / ID: 1
Name.synonym: gp19, large terminase, DNA-packaging protein B
Details: gp19 monomer 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 (E. coli) / Recombinant strain: BL21(DE3) 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
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 67000
Sample stageSpecimen holder: GATAN convetional holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: FFT live
DateOct 23, 2009
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 1005 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each plate
Final angle assignmentDetails: SPIDER
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.33 CUT-OFF / Software - Name: XMIPP, EMAN, Spider / Number images used: 3650
DetailsThe particles were selected using Maximum Likelihood procedures form an initial set of 14097 particles.

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera, SITUS
Details3CPE PDB was used as a template for the generation of the homology model of gp19 protein
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: volumetric correlation
Output model

PDB-4bij:
Threading model of T7 large terminase

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