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- EMDB-5695: Electron cryo-microscopy of human cytomegalovirus capsid -

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Entry
Database: EMDB / ID: EMD-5695
TitleElectron cryo-microscopy of human cytomegalovirus capsid
Map dataReconstruction of human cytomegalovirus (HCMV) capsids purified from cell nuclei
Sample
  • Sample: human cytomegalovirus (HCMV) capsid
  • Virus: Human herpesvirus 5
Keywordsherpesvirus / betaherpesvirus / human cytomegalovirus
Biological speciesHuman herpesvirus 5
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsDai XH / Yu XK / Gong H / Jiang XH / Abenes G / Liu HR / Shivakoti S / Britt W / Zhu H / Liu FY / Zhou ZH
CitationJournal: PLoS Pathog / Year: 2013
Title: The smallest capsid protein mediates binding of the essential tegument protein pp150 to stabilize DNA-containing capsids in human cytomegalovirus.
Authors: Xinghong Dai / Xuekui Yu / Hao Gong / Xiaohong Jiang / Gerrado Abenes / Hongrong Liu / Sakar Shivakoti / William J Britt / Hua Zhu / Fenyong Liu / Z Hong Zhou /
Abstract: Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes birth defects in newborns and life-threatening complications in immunocompromised individuals. Among all human herpesviruses, HCMV ...Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes birth defects in newborns and life-threatening complications in immunocompromised individuals. Among all human herpesviruses, HCMV contains a much larger dsDNA genome within a similarly-sized capsid compared to the others, and it was proposed to require pp150, a tegument protein only found in cytomegaloviruses, to stabilize its genome-containing capsid. However, little is known about how pp150 interacts with the underlying capsid. Moreover, the smallest capsid protein (SCP), while dispensable in herpes simplex virus type 1, was shown to play essential, yet undefined, role in HCMV infection. Here, by cryo electron microscopy (cryoEM), we determine three-dimensional structures of HCMV capsid (no pp150) and virion (with pp150) at sub-nanometer resolution. Comparison of these two structures reveals that each pp150 tegument density is composed of two helix bundles connected by a long central helix. Correlation between the resolved helices and sequence-based secondary structure prediction maps the tegument density to the N-terminal half of pp150. The structures also show that SCP mediates interactions between the capsid and pp150 at the upper helix bundle of pp150. Consistent with this structural observation, ribozyme inhibition of SCP expression in HCMV-infected cells impairs the formation of DNA-containing viral particles and reduces viral yield by 10,000 fold. By cryoEM reconstruction of the resulting "SCP-deficient" viral particles, we further demonstrate that SCP is required for pp150 functionally binding to the capsid. Together, our structural and biochemical results point to a mechanism whereby SCP recruits pp150 to stabilize genome-containing capsid for the production of infectious HCMV virion.
History
DepositionJun 22, 2013-
Header (metadata) releaseOct 16, 2013-
Map releaseOct 16, 2013-
UpdateOct 16, 2013-
Current statusOct 16, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 9.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 9.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 9.1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5695.tif

Downloads & links

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Map

FileDownload / File: emd_5695.map.gz / Format: CCP4 / Size: 976.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of human cytomegalovirus (HCMV) capsids purified from cell nuclei
Voxel sizeX=Y=Z: 2.152 Å
Density
Contour LevelBy AUTHOR: 9.1 / Movie #1: 9.1
Minimum - Maximum-10.41716385 - 30.544338230000001
Average (Standard dev.)0.4555279 (±2.69054842)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-320-320-320
Dimensions640640640
Spacing640640640
CellA=B=C: 1377.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1522.1522.152
M x/y/z640640640
origin x/y/z0.0000.0000.000
length x/y/z1377.2801377.2801377.280
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-320-320-320
NC/NR/NS640640640
D min/max/mean-10.41730.5440.456

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Supplemental data

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Sample components

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Entire : human cytomegalovirus (HCMV) capsid

EntireName: human cytomegalovirus (HCMV) capsid
Components
  • Sample: human cytomegalovirus (HCMV) capsid
  • Virus: Human herpesvirus 5

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Supramolecule #1000: human cytomegalovirus (HCMV) capsid

SupramoleculeName: human cytomegalovirus (HCMV) capsid / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Human herpesvirus 5

SupramoleculeName: Human herpesvirus 5 / type: virus / ID: 1 / Name.synonym: human cytomegalovirus / Details: purified from HCMV-infected cell nuclei / NCBI-ID: 10359 / Sci species name: Human herpesvirus 5 / Database: NCBI / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: Yes / Virus empty: No / Syn species name: human cytomegalovirus
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Virus shellShell ID: 1 / Diameter: 1350 Å / T number (triangulation number): 16

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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: 7.4
Details: Phosphate buffered saline (pH=7.4): 144 mg/L KH2PO4, 795 mg/L Na2HPO4-7H2O, 0.9% (w/w) NaCl
GridDetails: Quantifoil R2/1 Cu-200mesh grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot force = 0, blot time = 20s

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureAverage: 80 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification. Astigmatism correction was also carried out for each particle during the data processing.
DetailsParallel beam illumination
DateJun 1, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 1500 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMIRS / Number images used: 1

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