National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM104556
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM129245
United States
National Science Foundation (NSF, United States)
DBI 1920374
United States
Citation
Journal: Sci Adv / Year: 2022 Title: Subcellular organization of viral particles during maturation of nucleus-forming jumbo phage. Authors: Vorrapon Chaikeeratisak / Kanika Khanna / Katrina T Nguyen / MacKennon E Egan / Eray Enustun / Emily Armbruster / Jina Lee / Kit Pogliano / Elizabeth Villa / Joe Pogliano / Abstract: Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages are generally assumed to assemble randomly throughout the host cell cytoplasm. Here, we ...Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages are generally assumed to assemble randomly throughout the host cell cytoplasm. Here, we show that viral particles of nucleus-forming jumbo phage PhiPA3 assemble into a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to capsids, and these particles accumulate over time in a spherical pattern, with tails oriented inward and the heads outward to form bouquets at specific subcellular locations. Bouquets localize at the same fixed distance from the phage nucleus even when it is mispositioned, suggesting an active mechanism for positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phages, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.
Download / File: emd_25217.map.gz / Format: CCP4 / Size: 531.1 MB / Type: IMAGE STORED AS SIGNED BYTE
Annotation
Slice of a cryo-electron tomogram of PhiPA3-infected Pseudomonas aeruginosa cell at 70 mpi (Cell 3)
Voxel size
X=Y=Z: 22.46 Å
Density
Minimum - Maximum
-128.0 - 127.0
Average (Standard dev.)
32.027630000000002 (±8.504201)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
176
Dimensions
1280
1236
352
Spacing
1236
1280
352
Cell
A: 27760.559 Å / B: 28748.799 Å / C: 7905.92 Å α=β=γ: 90.0 °
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Supplemental data
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Sample components
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Entire : Pseudomonas aeruginosa infected with bacteriophage PhiPA3 at 75 mpi
Entire
Name: Pseudomonas aeruginosa infected with bacteriophage PhiPA3 at 75 mpi
Components
Cell: Pseudomonas aeruginosa infected with bacteriophage PhiPA3 at 75 mpi
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Supramolecule #1: Pseudomonas aeruginosa infected with bacteriophage PhiPA3 at 75 mpi
Supramolecule
Name: Pseudomonas aeruginosa infected with bacteriophage PhiPA3 at 75 mpi type: cell / ID: 1 / Parent: 0
Source (natural)
Organism: Pseudomonas aeruginosa PA1 (bacteria)
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Experimental details
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Structure determination
Method
cryo EM
Processing
electron tomography
Aggregation state
cell
-
Sample preparation
Buffer
pH: 7
Vitrification
Cryogen name: ETHANE-PROPANE
Sectioning
Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.03 / Focused ion beam - Duration: 1800 / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 2000 / Focused ion beam - Final thickness: 150 Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is FEI Scios. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.
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Electron microscopy
Microscope
FEI POLARA 300
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
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Image processing
Final reconstruction
Number images used: 60
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