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- EMDB-6330: 3D structure of the decameric assembly of the DNA-injection prote... -

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Basic information

Entry
Database: EMDB / ID: EMD-6330
Title3D structure of the decameric assembly of the DNA-injection protein gp12 from bacteriophage Sf6
Map dataEM reconstruction of DNA injection protein decameric assembly
Sample
  • Sample: gp12 of bacteriophage Sf6
  • Protein or peptide: product of gene 12
Keywordsbacteriophage / podovirus / DNA injection / decamer / Gram-negative / cell envelope
Function / homologyDNA transfer protein / DNA/protein translocase of phage P22 injectosome / Gene 12 protein
Function and homology information
Biological speciesBacillus phage SF6 (virus)
Methodsingle particle reconstruction / negative staining / Resolution: 11.0 Å
AuthorsZhao H / Speir JA / Matsui T / Lin Z / Liang L / Lynn AY / Varnado B / Weiss TM / Tang L
CitationJournal: PLoS One / Year: 2016
Title: Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel.
Authors: Haiyan Zhao / Jeffrey A Speir / Tsutomu Matsui / Zihan Lin / Lingfei Liang / Anna Y Lynn / Brittany Varnado / Thomas M Weiss / Liang Tang /
Abstract: The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we ...The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.
History
DepositionApr 30, 2015-
Header (metadata) releaseJun 17, 2015-
Map releaseMay 4, 2016-
UpdateMay 4, 2016-
Current statusMay 4, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6330.jpg

Downloads & links

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Map

FileDownload / File: emd_6330.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM reconstruction of DNA injection protein decameric assembly
Voxel sizeX=Y=Z: 2.73 Å
Density
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.11448574 - 0.08097367
Average (Standard dev.)0.00020927 (±0.0062018)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 349.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.732.732.73
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z349.440349.440349.440
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-31-64
NX/NY/NZ6963129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1140.0810.000

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Supplemental data

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Sample components

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Entire : gp12 of bacteriophage Sf6

EntireName: gp12 of bacteriophage Sf6
Components
  • Sample: gp12 of bacteriophage Sf6
  • Protein or peptide: product of gene 12

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Supramolecule #1000: gp12 of bacteriophage Sf6

SupramoleculeName: gp12 of bacteriophage Sf6 / type: sample / ID: 1000 / Details: The sample was mostly monodisperse. / Oligomeric state: decamer / Number unique components: 1
Molecular weightExperimental: 465 KDa / Theoretical: 465 KDa / Method: SDS PAGE and size exclusion

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Macromolecule #1: product of gene 12

MacromoleculeName: product of gene 12 / type: protein_or_peptide / ID: 1 / Name.synonym: gp12 / Number of copies: 10 / Oligomeric state: decamer / Recombinant expression: Yes
Source (natural)Organism: Bacillus phage SF6 (virus) / synonym: phage Sf6
Molecular weightExperimental: 46.5 KDa / Theoretical: 46.5 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET28b
SequenceUniProtKB: Gene 12 protein

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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

Concentration0.0415 mg/mL
BufferpH: 7.4 / Details: 20 mM NaPO4, 100 mM NaCl, 1 mM EDTA
StainingType: NEGATIVE
Details: 3 uL sample was applied onto a grid that was plasma cleaned with Ar/O2 mixture (20 sec, full power)
GridDetails: copper 400 mesh with carbon over vinyl
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -2.45 µm / Nominal defocus min: -0.29 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 90 K
DateAug 21, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 494 / Average electron dose: 38.89 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND
Final two d classificationNumber classes: 23
Final angle assignmentDetails: RELION: theta 180 degrees, phi 36 degrees
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 54104
DetailsA total of 86783 particles were boxed with a dimension of 128x128 pixels and a pixel size of 2.73 A using the program DoGPicker in the Appion pipeline. Image analysis and 3D reconstruction were performed using RELION. No symmetry was applied during 2D classification. After 2D classification, 54104 particles associated with high-quality class averages were included for the subsequent 3D reconstruction. In the 3D reconstruction, 10-fold rotational symmetry was applied.
FSC plot (resolution estimation)

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