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- EMDB-13665: PhiCPV4 bacteriophage Portal Protein -

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Basic information

Entry
Database: EMDB / ID: EMD-13665
TitlePhiCPV4 bacteriophage Portal Protein
Map data
Sample
  • Complex: PhiCPV4 phage portal protein
    • Protein or peptide: Connector protein
KeywordsBacteriophage / portal protein / cryoEM / VIRAL PROTEIN
Function / homologyPortal protein Gp10 / Phage Connector (GP10) / Portal protein Gp10 superfamily / Connector protein
Function and homology information
Biological speciesClostridium perfringens (bacteria) / Clostridium phage phiCPV4 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsJaved A / Villanueva H
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M009513 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R002622/1 United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: Viruses / Year: 2021
Title: Cryo-EM Structures of Two Bacteriophage Portal Proteins Provide Insights for Antimicrobial Phage Engineering.
Authors: Abid Javed / Hugo Villanueva / Shadikejiang Shataer / Sara Vasciaveo / Renos Savva / Elena V Orlova /
Abstract: Widespread antibiotic resistance has returned attention to bacteriophages as a means of managing bacterial pathogenesis. Synthetic biology approaches to engineer phages have demonstrated genomic ...Widespread antibiotic resistance has returned attention to bacteriophages as a means of managing bacterial pathogenesis. Synthetic biology approaches to engineer phages have demonstrated genomic editing to broaden natural host ranges, or to optimise microbicidal action. Gram positive pathogens cause serious pastoral animal and human infections that are especially lethal in newborns. Such pathogens are targeted by the obligate lytic phages of the and families. These phages have relatively small ~20 kb linear protein-capped genomes and their compact organisation, relatively few structural elements, and broad host range, are appealing from a phage-engineering standpoint. In this study, we focus on portal proteins, which are core elements for the assembly of such tailed phages. The structures of dodecameric portal complexes from phage GA1, which targets , and phage phiCPV4 that infects , were determined at resolutions of 3.3 Å and 2.9 Å, respectively. Both are found to closely resemble the related phi29 portal protein fold. However, the portal protein of phiCPV4 exhibits interesting differences in the clip domain. These structures provide new insights on structural diversity in portal proteins and will be essential for considerations in phage structural engineering.
History
DepositionOct 1, 2021-
Header (metadata) releaseOct 12, 2022-
Map releaseOct 12, 2022-
UpdateOct 25, 2023-
Current statusOct 25, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13665.png

Downloads & links

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Map

FileDownload / File: emd_13665.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.023
Minimum - Maximum-0.10513125 - 0.19952981
Average (Standard dev.)0.00015108797 (±0.004682096)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : PhiCPV4 phage portal protein

EntireName: PhiCPV4 phage portal protein
Components
  • Complex: PhiCPV4 phage portal protein
    • Protein or peptide: Connector protein

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Supramolecule #1: PhiCPV4 phage portal protein

SupramoleculeName: PhiCPV4 phage portal protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Dodecamer complex made up of 12 subunits.
Source (natural)Organism: Clostridium perfringens (bacteria)

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Macromolecule #1: Connector protein

MacromoleculeName: Connector protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Clostridium phage phiCPV4 (virus)
Molecular weightTheoretical: 34.652238 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKRKSYWNQ GDKILLKNDV FMVDRYYDYY SNMGLNRFRW KNLPPGMESR HIEQALFNEG QAVFFKNTDP NEPYGFLCLP CAPSNGQNI YGDPVDFNGI GVNKYFTNLS PLNAVRILDN DNGLAPVRHI AYYTYLMSQI EMTINMNLDQ QKFPIIIGAT Q KNKLSMEN ...String:
MSKRKSYWNQ GDKILLKNDV FMVDRYYDYY SNMGLNRFRW KNLPPGMESR HIEQALFNEG QAVFFKNTDP NEPYGFLCLP CAPSNGQNI YGDPVDFNGI GVNKYFTNLS PLNAVRILDN DNGLAPVRHI AYYTYLMSQI EMTINMNLDQ QKFPIIIGAT Q KNKLSMEN LYEKYSSFEP NILVDEKLAQ ALQEGKGFDA LNTQAPYLLD KLADFKKTCE NELLTFLGIN NTNNDKRERL LT DEVNANN SQITFVLEMA YKNRLDACKR INEMFGLNLE VEKVVNLLEV DMKGDVKNEG INGE

UniProtKB: Connector protein

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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

Concentration0.05 mg/mL
BufferpH: 7.5
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: 3 ul of sample was applied to lacey carbon grids..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 95.0 K / Max: 98.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-12 / Number grids imaged: 1 / Number real images: 2903 / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 89560
Startup modelType of model: OTHER
Details: Low-resolution 3D map calculated from negative stain images using RELION.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C12 (12 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 26940
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 74.6
Output model

PDB-7pv4:
PhiCPV4 bacteriophage Portal Protein

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