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- EMDB-5985: CryoEM of Bacteriophage PRD1 procapsid -

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Basic information

Entry
Database: EMDB / ID: EMD-5985
TitleCryoEM of Bacteriophage PRD1 procapsid
Map dataReconstruction of PRD1 procapsid without icosahedral symmetry imposition
Sample
  • Sample: Bacteriophage PRD1 procapsid
  • Virus: Bacteriophage PRD1 procapsid
KeywordsVirus / Genome packaging
Biological speciesBacteriophage PRD1 procapsid
Methodsingle particle reconstruction / cryo EM / Resolution: 14.0 Å
AuthorsHong C / Oksanen HM / Liu X / Jakana J / Bamford DH / Chiu W
CitationJournal: PLoS Biol / Year: 2014
Title: A structural model of the genome packaging process in a membrane-containing double stranded DNA virus.
Authors: Chuan Hong / Hanna M Oksanen / Xiangan Liu / Joanita Jakana / Dennis H Bamford / Wah Chiu /
Abstract: Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the ...Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.
History
DepositionJun 10, 2014-
Header (metadata) releaseJul 30, 2014-
Map releaseMay 20, 2015-
UpdateMay 20, 2015-
Current statusMay 20, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5985.gif

Downloads & links

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Map

FileDownload / File: emd_5985.map.gz / Format: CCP4 / Size: 804.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of PRD1 procapsid without icosahedral symmetry imposition
Voxel sizeX=Y=Z: 1.42 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-3.2746191 - 2.33735561
Average (Standard dev.)0.14314391 (±0.38011107)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-300-300-300
Dimensions600600600
Spacing600600600
CellA=B=C: 852.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.421.421.42
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z852.000852.000852.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-300-300-300
NC/NR/NS600600600
D min/max/mean-3.2752.3370.143

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Supplemental data

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Sample components

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Entire : Bacteriophage PRD1 procapsid

EntireName: Bacteriophage PRD1 procapsid
Components
  • Sample: Bacteriophage PRD1 procapsid
  • Virus: Bacteriophage PRD1 procapsid

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Supramolecule #1000: Bacteriophage PRD1 procapsid

SupramoleculeName: Bacteriophage PRD1 procapsid / type: sample / ID: 1000 / Details: The genome DNA was not packaged. / Oligomeric state: Icosahedral Virus / Number unique components: 1
Molecular weightExperimental: 66 MDa / Theoretical: 66 MDa / Method: Sedimentation

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Supramolecule #1: Bacteriophage PRD1 procapsid

SupramoleculeName: Bacteriophage PRD1 procapsid / type: virus / ID: 1 / Details: Packaging deficient procapsid / Sci species name: Bacteriophage PRD1 procapsid / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
synonym: BACTERIA(EUBACTERIA)
Host systemOrganism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightExperimental: 66 MDa / Theoretical: 66 MDa
Virus shellShell ID: 1 / Diameter: 650 Å / T number (triangulation number): 25

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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

Concentration10 mg/mL
BufferpH: 7.2 / Details: 20 mM potassium phosphate, 1 mM MgCl2
GridDetails: 400 mesh 1.2/1.3, plasma-cleaned
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 2 seconds before plunging.

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 106000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.1 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000
Specialist opticsEnergy filter - Name: JEOL Omega / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification.
DateAug 21, 2009
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 340 / Average electron dose: 20 e/Å2 / Camera length: 150

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Image processing

CTF correctionDetails: Each frame
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: OTHER / Software - Name: MPSA, EMAN, EMAN2 / Number images used: 4300
DetailsNo icosahedral symmetry was imposed.

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Atomic model buiding 1

Initial modelPDB ID:

1w8x

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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