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- PDB-8cez: HK97 Portal Protein In situ (prohead II) -

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Basic information

Entry
Database: PDB / ID: 8cez
TitleHK97 Portal Protein In situ (prohead II)
ComponentsPortal protein
KeywordsVIRAL PROTEIN / portal / HK97 / bacteriophage / packaging
Function / homologyPhage portal protein, HK97 / Bacteriophage/Gene transfer agent portal protein / Phage portal protein / viral capsid / Portal protein
Function and homology information
Biological speciesHendrixvirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsHawkins, D.E.D.P. / Antson, A.A.
Funding support United Kingdom, 1items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 24, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 2, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Portal protein
B: Portal protein
C: Portal protein
D: Portal protein
E: Portal protein
F: Portal protein
G: Portal protein
H: Portal protein
I: Portal protein
J: Portal protein
K: Portal protein
L: Portal protein


Theoretical massNumber of molelcules
Total (without water)567,82212
Polymers567,82212
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Portal protein


Mass: 47318.488 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hendrixvirus / Production host: Escherichia phage EcSzw-2 (virus) / References: UniProt: Q77W97

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Hendrixvirus / Type: VIRUS
Details: Proheads were produced by infection of E. coli 594 cells with HK97 amber mutant amC2, propagated using Escherichia LE392 cells.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.56 MDa / Experimental value: NO
Source (natural)Organism: Hendrixvirus / Strain: HK97
Source (recombinant)Organism: Escherichia phage EcSzw-2 (virus) / Strain: LE392 / Cell: E.coli
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Escherichia phage EcSzw-2 / Strain: 594
Buffer solutionpH: 7.5
Details: final buffer = 20 mM Tris pH 7.5, 10 mM MgCl2, 50 mM KGlu, 5 mM ATP-gamma-S
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Packaging 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.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2RELION3.1image acquisition
4RELION3.1CTF correction
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 82279
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57279 / Num. of class averages: 3 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00335016
ELECTRON MICROSCOPYf_angle_d0.59447268
ELECTRON MICROSCOPYf_dihedral_angle_d6.0984752
ELECTRON MICROSCOPYf_chiral_restr0.045124
ELECTRON MICROSCOPYf_plane_restr0.0056216

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