|Entry||Database: EMDB / ID: 4445|
|Title||Thermophage P23-45 procapsid asymmetric reconstruction|
|Map data||Thermophage P23-45 procapsid asymmetric reconstruction|
|Sample||Thermus phage P2345:|
virus / Major head protein / Portal protein
|Source||Thermus phage P2345 (bacteriophage)|
|Method||single particle reconstruction / cryo EM / 9.33 Å resolution|
|Authors||Bayfield OW / Klimuk E / Winkler DC / Hesketh EL / Chechik M / Cheng N / Dykeman EC / Minakhin L / Ranson NA / Severinov K / Steven AC / Antson AA|
|Citation||Journal: 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.
|Date||Deposition: Nov 29, 2018 / Header (metadata) release: Feb 13, 2019 / Map release: Feb 13, 2019 / Last update: Mar 13, 2019|
|Structure viewer||EM map: |
Downloads & links
|File||emd_4445.map.gz (map file in CCP4 format, 1088392 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.5975 Å|
CCP4 map header:
-Entire Thermus phage P2345
|Entire||Name: Thermus phage P2345 / Number of components: 3|
-Component #1: virus, Thermus phage P2345
|Virus||Name: Thermus phage P2345 / Class: VIRION / Empty: Yes / Enveloped: No / Isolate: SPECIES|
|Species||Species: Thermus phage P2345 (bacteriophage)|
|Source (natural)||Host Species: Thermus thermophilus HB8 (bacteria)|
-Component #2: protein, Major head protein
|Protein||Name: Major head protein / Recombinant expression: No|
|Source||Species: Thermus phage P2345 (bacteriophage)|
-Component #3: protein, Portal protein
|Protein||Name: Portal protein / Recombinant expression: No|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||pH: 8|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 283 K / Humidity: 90 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 99 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 75000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 2500.0 nm|
|Specimen Holder||Model: OTHER|
|Camera||Detector: FEI FALCON III (4k x 4k)|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 16758|
|3D reconstruction||Software: RELION / Resolution: 9.33 Å / Resolution method: FSC 0.143 CUT-OFF|
-Atomic model buiding
|Modeling #1||Refinement space: REAL|
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