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- EMDB-4447: Thermophage P23-45 procapsid icosahedral reconstruction -

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Basic information

Entry
Database: EMDB / ID: EMD-4447
TitleThermophage P23-45 procapsid icosahedral reconstruction
Map dataThermophage P23-45 procapsid icosahedral reconstruction
Sample
  • Virus: Thermus phage P2345 (virus)
    • Protein or peptide: Major head protein
Keywordsbacteriophage / thermophage / caudovirales / siphoviridae / capsid / procapsid / auxiliary / HK97 / virus
Function / homologyMajor capsid protein GpE / Phage major capsid protein E / T=7 icosahedral viral capsid / host cell cytoplasm / Major capsid protein
Function and homology information
Biological speciesThermus virus P23-45 / Thermus phage P2345 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.39 Å
AuthorsBayfield OW / Klimuk E / Winkler DC / Hesketh EL / Chechik M / Cheng N / Dykeman EC / Minakhin L / Ranson NA / Severinov K ...Bayfield OW / Klimuk E / Winkler DC / Hesketh EL / Chechik M / Cheng N / Dykeman EC / Minakhin L / Ranson NA / Severinov K / Steven AC / Antson AA
Funding support United Kingdom, United States, Russian Federation, 6 items
OrganizationGrant numberCountry
Wellcome Trust206377 United Kingdom
Wellcome Trust103460 United Kingdom
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS) 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.
History
DepositionNov 29, 2018-
Header (metadata) releaseJan 2, 2019-
Map releaseFeb 13, 2019-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ibc
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ibc
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4447.png

Downloads & links

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Map

FileDownload / File: emd_4447.map.gz / Format: CCP4 / Size: 1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThermophage P23-45 procapsid icosahedral reconstruction
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.6 Å/pix.
x 648 pix.
= 1035.18 Å		1.6 Å/pix.
x 648 pix.
= 1035.18 Å		1.6 Å/pix.
x 648 pix.
= 1035.18 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5975 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.051979132 - 0.20229945
Average (Standard dev.)-0.00015836759 (±0.012459133)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions648648648
Spacing648648648
CellA=B=C: 1035.1799 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.59751.59751.5975
M x/y/z648648648
origin x/y/z0.0000.0000.000
length x/y/z1035.1801035.1801035.180
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS648648648
D min/max/mean-0.0520.202-0.000

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Supplemental data

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Sample components

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Entire : Thermus phage P2345

EntireName: Thermus phage P2345 (virus)
Components
  • Virus: Thermus phage P2345 (virus)
    • Protein or peptide: Major head protein

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Supramolecule #1: Thermus phage P2345

SupramoleculeName: Thermus phage P2345 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 466051 / Sci species name: Thermus phage P2345 / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 19.58 MDa
Virus shellShell ID: 1 / T number (triangulation number): 7

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Macromolecule #1: Major head protein

MacromoleculeName: Major head protein / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Thermus virus P23-45
Molecular weightTheoretical: 46.680754 KDa
SequenceString: MRVPININNA LARVRDPLSI GGLKFPTTKE IQEAVAAIAD KFNQENDLVD RFFPEDSTFA SELELYLLRT QDAEQTGMTF VHQVGSTSL PVEARVAKVD LAKATWSPLA FKESRVWDEK EILYLGRLAD EVQAGVINEQ IAESLTWLMA RMRNRRRWLT W QVMRTGRI ...String:
MRVPININNA LARVRDPLSI GGLKFPTTKE IQEAVAAIAD KFNQENDLVD RFFPEDSTFA SELELYLLRT QDAEQTGMTF VHQVGSTSL PVEARVAKVD LAKATWSPLA FKESRVWDEK EILYLGRLAD EVQAGVINEQ IAESLTWLMA RMRNRRRWLT W QVMRTGRI TIQPNDPYNP NGLKYVIDYG VTDIELPLPQ KFDAKDGNGN SAVDPIQYFR DLIKAATYFP DRRPVAIIVG PG FDEVLAD NTFVQKYVEY EKGWVVGQNT VQPPREVYRQ AALDIFKRYT GLEVMVYDKT YRDQDGSVKY WIPVGELIVL NQS TGPVGR FVYTAHVAGQ RNGKVVYATG PYLTVKDHLQ DDPPYYAIIA GFHGLPQLSG YNTEDFSFHR FKWLKYANNV QSYL PPFPP KVEL

UniProtKB: Major capsid 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: 8
Component:
ConcentrationFormula
100.0 mMNaCl
20.0 mMTris-HCl
10.0 mMMgCL2
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 2.0 sec. / Average electron dose: 99.0 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: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 75000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 4.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 38044
Initial angle assignmentType: OTHER / Software - Name: RELION
Final angle assignmentType: OTHER / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6ibc:
Thermophage P23-45 procapsid

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