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

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Basic information

Entry
Database: EMDB / ID: EMD-4445
TitleThermophage P23-45 procapsid asymmetric reconstruction
Map dataThermophage P23-45 procapsid asymmetric reconstruction
Sample
  • Virus: Thermus phage P2345 (virus)
    • Protein or peptide: Major head protein
    • Protein or peptide: Portal protein
Biological speciesThermus phage P2345 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.33 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
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases United States
Wellcome Trust103460 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/L021250/1 United Kingdom
Russian Foundation for Basic Research16-34-60137 Russian Federation
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) releaseFeb 13, 2019-
Map releaseFeb 13, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • 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

FileDownload / File: emd_4445.map.gz / Format: CCP4 / Size: 1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThermophage P23-45 procapsid asymmetric reconstruction
Voxel sizeX=Y=Z: 1.5975 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.09
Minimum - Maximum-0.08378945 - 0.1804991
Average (Standard dev.)0.000109451816 (±0.018017964)
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-0.0840.1800.000

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Supplemental data

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Sample components

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Entire : Thermus phage P2345

EntireName: Thermus phage P2345 (virus)
Components
  • Virus: Thermus phage P2345 (virus)
    • Protein or peptide: Major head protein
    • Protein or peptide: Portal 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)
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 / Enantiomer: LEVO
Source (natural)Organism: Thermus phage P2345 (virus)
SequenceString: MRVPININNA LARVRDPLSI GGLKFPTTKE IQEAVAAIAD KFNQENDLVD RFFPEDSTFA SELELYLLRT QDAEQTGMTF VHQVGSTSLP VEARVAKVDL AKATWSPLAF KESRVWDEKE ILYLGRLADE VQAGVINEQI AESLTWLMAR MRNRRRWLTW QVMRTGRITI ...String:
MRVPININNA LARVRDPLSI GGLKFPTTKE IQEAVAAIAD KFNQENDLVD RFFPEDSTFA SELELYLLRT QDAEQTGMTF VHQVGSTSLP VEARVAKVDL AKATWSPLAF KESRVWDEKE ILYLGRLADE VQAGVINEQI AESLTWLMAR MRNRRRWLTW QVMRTGRITI QPNDPYNPNG LKYVIDYGVT DIELPLPQKF DAKDGNGNSA VDPIQYFRDL IKAATYFPDR RPVAIIVGPG FDEVLADNTF VQKYVEYEKG WVVGQNTVQP PREVYRQAAL DIFKRYTGLE VMVYDKTYRD QDGSVKYWIP VGELIVLNQS TGPVGRFVYT AHVAGQRNGK VVYATGPYLT VKDHLQDDPP YYAIIAGFHG LPQLSGYNTE DFSFHRFKWL KYANNVQSYL PPFPPKVEL

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Macromolecule #2: Portal protein

MacromoleculeName: Portal protein / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
SequenceString: MAKRGRKPKE LVPGPGSIDP SDVPKLEGAS VPVMSTSYDV VVDREFDELL QGKDGLLVYH KMLSDGTVKN ALNYIFGRIR SAKWYVEPAS TDPEDIAIAA FIHAQLGIDD ASVGKYPFGR LFAIYENAYI YGMAAGEIVL TLGADGKLIL DKIVPIHPFN IDEVLYDEEG ...String:
MAKRGRKPKE LVPGPGSIDP SDVPKLEGAS VPVMSTSYDV VVDREFDELL QGKDGLLVYH KMLSDGTVKN ALNYIFGRIR SAKWYVEPAS TDPEDIAIAA FIHAQLGIDD ASVGKYPFGR LFAIYENAYI YGMAAGEIVL TLGADGKLIL DKIVPIHPFN IDEVLYDEEG GPKALKLSGE VKGGSQFVNG LEIPIWKTVV FLHNDDGSFT GQSALRAAVP HWLAKRALIL LINHGLERFM IGVPTLTIPK SVRQGTKQWE AAKEIVKNFV QKPRHGIILP DDWKFDTVDL KSAMPDAIPY LTYHDAGIAR ALGIDFNTVQ LNMGVQAVNI GEFVSLTQQT IISLQREFAS AVNLYLIPKL VLPNWPGATR FPRLTFEMEE RNDFSAAANL MGMLINAVKD SEDIPTELKA LIDALPSKMR RALGVVDEVR EAVRQPADSR YLYTRRRR

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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 mMNaClSodium chloride
20.0 mMTris-HClTris
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 75000
Sample stageCooling holder cryogen: NITROGEN
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: OTHER / Software - Name: RELION
Final angle assignmentType: OTHER / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.33 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 16758
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL

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