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- PDB-6i9e: Thermophage P23-45 empty expanded capsid -

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Basic information

Entry
Database: PDB / ID: 6i9e
TitleThermophage P23-45 empty expanded capsid
Components
  • Auxiliary protein
  • Major head protein
KeywordsVIRUS / bacteriophage / thermophage / caudovirales / siphoviridae / capsid / auxiliary / HK97
Function / homologyPhage major capsid protein E / Major capsid protein GpE / viral capsid, decoration / T=7 icosahedral viral capsid / host cell cytoplasm / Decoration protein / Major capsid protein
Function and homology information
Biological speciesThermus virus P23-45 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.74 Å
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.
Funding support United Kingdom, United States, Russian Federation, 6items
OrganizationGrant numberCountry
Wellcome Trust206377 United Kingdom
Wellcome Trust103460 United Kingdom
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases United States
Russian Foundation for Basic Research16-34-60137 Russian Federation
Biotechnology and Biological Sciences Research CouncilBB/L021250/1 United Kingdom
Wellcome Trust108466 United Kingdom
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 ...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
History
DepositionNov 23, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 6, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Data collection / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc / pdbx_struct_assembly_gen / pdbx_struct_oper_list
Item: _citation.country / _citation.journal_abbrev ..._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 / _pdbx_struct_assembly_gen.oper_expression
Revision 1.2Mar 6, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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  • Deposited structure unit
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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
H: Auxiliary protein
I: Auxiliary protein
J: Auxiliary protein
K: Auxiliary protein
L: Auxiliary protein
M: Auxiliary protein
N: Auxiliary protein


Theoretical massNumber of molelcules
Total (without water)443,39214
Polymers443,39214
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
H: Auxiliary protein
I: Auxiliary protein
J: Auxiliary protein
K: Auxiliary protein
L: Auxiliary protein
M: Auxiliary protein
N: Auxiliary protein
x 60


Theoretical massNumber of molelcules
Total (without water)26,603,538840
Polymers26,603,538840
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation59
Buried area65050 Å2
ΔGint-359 kcal/mol
Surface area193160 Å2
MethodPISA

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Components

#1: Protein
Major head protein


Mass: 46680.754 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Thermus virus P23-45 (bacteriophage) / Cell line: Thermus thermophilus HB8 / References: UniProt: A7XXC2
#2: Protein
Auxiliary protein


Mass: 16661.002 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Thermus virus P23-45 (bacteriophage) / Cell line: Thermus thermophilus HB8 / References: UniProt: A7XXC1

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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: Thermus phage P2345 / Type: VIRUS / Entity ID: 1, 2 / Source: NATURAL
Molecular weightValue: 26.57 MDa / Experimental value: NO
Source (natural)Organism: Thermus phage P2345 (bacteriophage)
Details of virusEmpty: YES / Enveloped: NO / Isolate: SPECIES / Type: VIRION
Natural hostOrganism: Thermus thermophilus HB8
Virus shellTriangulation number (T number): 7
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
1100 mMNaClSodium chloride1
220 mMTris-HClTris1
310 mMMgCL21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
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
9PHENIXmodel refinement
10Cootmodel refinement
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2372 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementHighest resolution: 3.74 Å
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00732284
ELECTRON MICROSCOPYf_angle_d0.90444044
ELECTRON MICROSCOPYf_dihedral_angle_d10.4419152
ELECTRON MICROSCOPYf_chiral_restr0.064739
ELECTRON MICROSCOPYf_plane_restr0.0085761

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