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- PDB-6ibc: Thermophage P23-45 procapsid -

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Entry
Database: PDB / ID: 6ibc
TitleThermophage P23-45 procapsid
ComponentsMajor head protein
KeywordsVIRUS / bacteriophage / thermophage / caudovirales / siphoviridae / capsid / procapsid / auxiliary / HK97 / virus
Function / homologyMajor capsid protein GpE / Phage major capsid protein E / Major head protein
Function and homology information
Specimen sourceThermus virus P23-45 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.39 Å resolution
AuthorsBayfield, O.W. / Klimuk, E. / Winkler, D.C. / Hesketh, E.L. / Chechik, M. / Cheng, N. / Dykeman, E.C. / Minakhin, L. / Ranson, N.A. / Severinov, K. / Steven, A.C. / Antson, A.A.
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019
Title: Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids.
Authors: Oliver W Bayfield / Evgeny Klimuk / Dennis C Winkler / Emma L Hesketh / Maria Chechik / Naiqian Cheng / Eric C Dykeman / Leonid Minakhin / Neil A Ranson / Konstantin Severinov / Alasdair C Steven / Alfred A Antson
Abstract: Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic ...Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 29, 2018 / Release: Feb 13, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Feb 13, 2019Structure modelrepositoryInitial release
1.1Feb 20, 2019Structure modelData collection / Database referencescitation / citation_author / em_admin / pdbx_database_proc_citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update
1.2Mar 6, 2019Structure modelData collection / Database referencescitation / citation_author / em_admin / pdbx_database_proc_citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
  • Imaged by Jmol
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-4447
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  • Superimposition on EM map
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Movie viewer
Structure viewerMolecule:
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Assembly

Deposited unit
A: Major head protein
B: Major head protein
C: Major head protein
D: Major head protein
E: Major head protein
F: Major head protein
G: Major head protein


Theoretical massNumber of molelcules
Total (without water)326,7657
Polyers326,7657
Non-polymers00
Water0
1
A: Major head protein
B: Major head protein
C: Major head protein
D: Major head protein
E: Major head protein
F: Major head protein
G: Major head protein
x 60


Theoretical massNumber of molelcules
Total (without water)19,605,917420
Polyers19,605,917420
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: Major head protein
B: Major head protein
C: Major head protein
D: Major head protein
E: Major head protein
F: Major head protein
G: Major head protein
x 5


  • icosahedral pentamer
  • 1.63 MDa, 35 polymers
Theoretical massNumber of molelcules
Total (without water)1,633,82635
Polyers1,633,82635
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: Major head protein
B: Major head protein
C: Major head protein
D: Major head protein
E: Major head protein
F: Major head protein
G: Major head protein
x 6


  • icosahedral 23 hexamer
  • 1.96 MDa, 42 polymers
Theoretical massNumber of molelcules
Total (without water)1,960,59242
Polyers1,960,59242
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1

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Components

#1: Protein/peptide
Major head protein


Mass: 46680.754 Da / Num. of mol.: 7 / Source: (natural) Thermus virus P23-45 (bacteriophage) / Cell line: Thermus thermophilus HB8 / References: UniProt: A7XXC2

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Thermus phage P2345 / Type: VIRUS / Entity ID: 1 / Source: NATURAL
Molecular weightValue: 19.58 MDa / Experimental value: NO
Source (natural)Organism: Thermus phage P2345 (bacteriophage)
Details of virusEmpty: YES / Enveloped: NO / Virus isolate: SPECIES / Virus type: VIRION
Natural hostOrganism: Thermus thermophilus HB8
Virus shellTriangulation number (T number): 7
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer ID
1100 mMNaCl1
220 mMTris-HCl1
310 mMMgCL21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 microns
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2 sec. / Electron dose: 99 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4CTFFINDCTF correction
7PHENIXmodel fitting
8Cootmodel fitting
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I
3D reconstructionResolution: 4.39 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 38044 / Symmetry type: POINT
Atomic model buildingRef protocol: AB INITIO MODEL / Ref space: REAL
Refine
Refine IDHighest resolution
14.39
ELECTRON MICROSCOPY
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00720273
ELECTRON MICROSCOPYf_angle_d1.05927690
ELECTRON MICROSCOPYf_dihedral_angle_d8.72811902
ELECTRON MICROSCOPYf_chiral_restr0.0563005
ELECTRON MICROSCOPYf_plane_restr0.0083659

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