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- EMDB-4445: Thermophage P23-45 procapsid asymmetric reconstruction -

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Basic information

Entry
Database: EMDB / ID: 4445
TitleThermophage P23-45 procapsid asymmetric reconstruction
Map dataThermophage P23-45 procapsid asymmetric reconstruction
SampleThermus phage P2345:
virus / Major head protein / Portal protein
SourceThermus phage P2345 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / 9.33 Å resolution
AuthorsBayfield OW / Klimuk E / Winkler DC / Hesketh EL / Chechik M / Cheng N / Dykeman EC / Minakhin L / Ranson NA / Severinov K / Steven AC / Antson AA
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.
DateDeposition: Nov 29, 2018 / Header (metadata) release: Feb 13, 2019 / Map release: Feb 13, 2019 / Last update: Mar 13, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.09
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_4445.map.gz (map file in CCP4 format, 1088392 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
648 pix
1.6 Å/pix.
= 1035.18 Å
648 pix
1.6 Å/pix.
= 1035.18 Å
648 pix
1.6 Å/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
Contour Level:0.05 (by author), 0.09 (movie #1):
Minimum - Maximum-0.08378945 - 0.1804991
Average (Standard dev.)0.000109451816 (0.018017964)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions648648648
Origin0.00.00.0
Limit647.0647.0647.0
Spacing648648648
CellA=B=C: 1035.1799 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ-205-205-205
NX/NY/NZ411411411
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS648648648
D min/max/mean-0.0840.1800.000

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Supplemental data

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Sample components

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Entire Thermus phage P2345

EntireName: Thermus phage P2345 / Number of components: 3

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Component #1: virus, Thermus phage P2345

VirusName: Thermus phage P2345 / Class: VIRION / Empty: Yes / Enveloped: No / Isolate: SPECIES
SpeciesSpecies: Thermus phage P2345 (bacteriophage)
Source (natural)Host Species: Thermus thermophilus HB8 (bacteria)

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Component #2: protein, Major head protein

ProteinName: Major head protein / Recombinant expression: No
SourceSpecies: Thermus phage P2345 (bacteriophage)

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Component #3: protein, Portal protein

ProteinName: Portal protein / Recombinant expression: No

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 283 K / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 99 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 2500.0 nm
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 16758
3D reconstructionSoftware: RELION / Resolution: 9.33 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL

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