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- EMDB-6429: Architecture of the complex formed by large and small Terminase s... -

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Basic information

Entry
Database: EMDB / ID: EMD-6429
TitleArchitecture of the complex formed by large and small Terminase subunits from Bacteriophage P22
Map dataAsymmetric reconstruction of terminase holoenzyme from Bacteriophage P22
Sample
  • Sample: Bacteriophage P22 terminase holoenzyme
  • Protein or peptide: small terminase subunit
  • Protein or peptide: large terminase subunit
Keywordsviral genome-packaging motor / Terminase complex / Salmonella virus / P22 Bacteriophage
Biological speciesEnterobacteria phage P22 (virus)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsMcNulty R / Johnson JE
CitationJournal: J Mol Biol / Year: 2015
Title: Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22.
Authors: Reginald McNulty / Ravi Kumar Lokareddy / Ankoor Roy / Yang Yang / Gabriel C Lander / Albert J R Heck / John E Johnson / Gino Cingolani /
Abstract: Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit ...Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1-58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140-162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a "nutcracker" with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.
History
DepositionAug 17, 2015-
Header (metadata) releaseSep 9, 2015-
Map releaseSep 9, 2015-
UpdateOct 14, 2015-
Current statusOct 14, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6429.png

Downloads & links

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Map

FileDownload / File: emd_6429.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric reconstruction of terminase holoenzyme from Bacteriophage P22
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.02977208 - 0.0939173
Average (Standard dev.)0.00002503 (±0.00756671)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 295.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z295.200295.200295.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-180
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-0.0300.0940.000

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Supplemental data

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Segmentation: Mask of complex generated automatically with RELION

AnnotationMask of complex generated automatically with RELION
Fileemd_6429_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 P22 terminase holoenzyme

EntireName: Bacteriophage P22 terminase holoenzyme
Components
  • Sample: Bacteriophage P22 terminase holoenzyme
  • Protein or peptide: small terminase subunit
  • Protein or peptide: large terminase subunit

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Supramolecule #1000: Bacteriophage P22 terminase holoenzyme

SupramoleculeName: Bacteriophage P22 terminase holoenzyme / type: sample / ID: 1000
Oligomeric state: One nonamer of S-terminase binds to two L-terminase subunits
Number unique components: 2
Molecular weightExperimental: 287 KDa / Theoretical: 287 KDa / Method: Native MS

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Macromolecule #1: small terminase subunit

MacromoleculeName: small terminase subunit / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Oligomeric state: 9 small subunits : 2 large subunits / Recombinant expression: Yes
Source (natural)Organism: Enterobacteria phage P22 (virus) / synonym: P22
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL-21-AI / Recombinant plasmid: pET30

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Macromolecule #2: large terminase subunit

MacromoleculeName: large terminase subunit / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Oligomeric state: 9 small subunits : 2 large subunits / Recombinant expression: Yes
Source (natural)Organism: Enterobacteria phage P22 (virus) / synonym: P22
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL-21-AI / Recombinant plasmid: pET30

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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

Concentration0.014 mg/mL
BufferpH: 8
Details: 20 mM Tris-HCl, 150 mM NaCl, 3 mm DTT, 5% glycerol, 1 mM MgCl2
StainingType: NEGATIVE
Details: Protein was adsorbed to the grid for 1 minute, blotted, and passed through four consecutive 40 microliter drops of 2% uranyl formate.
GridDetails: 400 mesh copper grids charged with Gatan plasma cleaner
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 0.2 µm / Nominal defocus min: 0.1 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateMar 25, 2015
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 44 / Average electron dose: 20 e/Å2 / Details: Images were acquired using Leginon.

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 2062
DetailsParticles were selected and filtered using the Appion Suite. Refinement was done with RELION.
FSC plot (resolution estimation)

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