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- EMDB-16614: HK97 Portal Protein In situ (prohead II) -

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Basic information

Entry
Database: EMDB / ID: EMD-16614
TitleHK97 Portal Protein In situ (prohead II)
Map dataHK97 bacteriophage portal protein in prohead II as part of packaging complex. Symmetry = C12.
Sample
  • Virus: Hendrixvirus
    • Protein or peptide: Portal protein
Keywordsportal / HK97 / bacteriophage / packaging / VIRAL PROTEIN
Function / homologyPhage portal protein, HK97 / Bacteriophage/Gene transfer agent portal protein / Phage portal protein / viral capsid / Portal protein
Function and homology information
Biological speciesHendrixvirus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsHawkins DEDP / Antson AA
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)AC014501 United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Insights into a viral motor: the structure of the HK97 packaging termination assembly.
Authors: Dorothy E D P Hawkins / Oliver W Bayfield / Herman K H Fung / Daniel N Grba / Alexis Huet / James F Conway / Alfred A Antson /
Abstract: Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each ...Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.
History
DepositionFeb 2, 2023-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateAug 2, 2023-
Current statusAug 2, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16614.png

Downloads & links

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Map

FileDownload / File: emd_16614.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHK97 bacteriophage portal protein in prohead II as part of packaging complex. Symmetry = C12.
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.27413908 - 0.43618548
Average (Standard dev.)0.0003557221 (±0.016429665)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 428.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16614_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: HK97 bacteriophage portal protein in prohead II as...

Fileemd_16614_half_map_1.map
AnnotationHK97 bacteriophage portal protein in prohead II as part of packaging complex. Symmetry = C12.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: HK97 bacteriophage portal protein in prohead II as...

Fileemd_16614_half_map_2.map
AnnotationHK97 bacteriophage portal protein in prohead II as part of packaging complex. Symmetry = C12.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hendrixvirus

EntireName: Hendrixvirus
Components
  • Virus: Hendrixvirus
    • Protein or peptide: Portal protein

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Supramolecule #1: Hendrixvirus

SupramoleculeName: Hendrixvirus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Proheads were produced by infection of E. coli 594 cells with HK97 amber mutant amC2, propagated using Escherichia LE392 cells.
NCBI-ID: 2169654 / Sci species name: Hendrixvirus / Sci species strain: HK97 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Escherichia phage EcSzw-2 (virus) / Strain: 594
Molecular weightTheoretical: 560 KDa

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Macromolecule #1: Portal protein

MacromoleculeName: Portal protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Hendrixvirus
Molecular weightTheoretical: 47.318488 KDa
Recombinant expressionOrganism: Escherichia phage EcSzw-2 (virus)
SequenceString: MEEPKYTIDL RTNNGWWARL KSWFVGGRLV TPNQGSQTGP VSAHGYLGDS SINDERILQI STVWRCVSLI STLTACLPLD VFETDQNDN RKKVDLSNPL ARLLRYSPNQ YMTAQEFREA MTMQLCFYGN AYALVDRNSA GDVISLLPLQ SANMDVKLVG K KVVYRYQR ...String:
MEEPKYTIDL RTNNGWWARL KSWFVGGRLV TPNQGSQTGP VSAHGYLGDS SINDERILQI STVWRCVSLI STLTACLPLD VFETDQNDN RKKVDLSNPL ARLLRYSPNQ YMTAQEFREA MTMQLCFYGN AYALVDRNSA GDVISLLPLQ SANMDVKLVG K KVVYRYQR DSEYADFSQK EIFHLKGFGF TGLVGLSPIA FACKSAGVAV AMEDQQRDFF ANGAKSPQIL STGEKVLTEQ QR SQVEENF KEIAGGPVKK RLWILEAGFS TSAIGVTPQD AEMMASRKFQ VSELARFFGV PPHLVGDVEK STSWGSGIEQ QNL GFLQYT LQPYISRWEN SIQRWLIPSK DVGRLHAEHN LDGLLRGDSA SRAAFMKAMG ESGLRTINEM RRTDNMPPLP GGDV AMRQA QYVPITDLGT NKEPRNNGA

UniProtKB: Portal 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

BufferpH: 7.5
Details: final buffer = 20 mM Tris pH 7.5, 10 mM MgCl2, 50 mM KGlu, 5 mM ATP-gamma-S
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.2 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV
DetailsPackaging reactions 30 nM hk97 proheads, 2.5 micromolar large terminase, 5 micromolar small terminase and 30 nM DNA, 75 micromolar ATP were incubated for 2 mins then 5 micromolar ATP-gamma-S was added.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 82279
Startup modelType of model: NONE
Final reconstructionNumber classes used: 3 / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 57279
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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