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- EMDB-3270: Importin-beta can bind Hepatitis B Virus core protein and empty c... -

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Entry
Database: EMDB / ID: EMD-3270
TitleImportin-beta can bind Hepatitis B Virus core protein and empty core-like particles and induce structural changes
Map data
SampleName:11 uM Cp183 dimer in capsid form and 18.8 uM Imp-beta in ammonium formate (dark particles):
virus / importin betaImportin
KeywordsHBV / Cp183 / Importin-beta
Biological speciesHepatitis B virus (HBV) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.8 Å
AuthorsChen C / Wang JC-Y / Pierson EE / Kiefer DZ / Delaleau M / Gallucci L / Cazenave C / Kann M / Jarrold MF / Zlotnick A
CitationJournal: PLoS Pathog / Year: 2016
Title: Importin β Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes.
Authors: Chao Chen / Joseph Che-Yen Wang / Elizabeth E Pierson / David Z Keifer / Mildred Delaleau / Lara Gallucci / Christian Cazenave / Michael Kann / Martin F Jarrold / Adam Zlotnick /
Abstract: Hepatitis B virus (HBV) capsids are found in many forms: immature single-stranded RNA-filled cores, single-stranded DNA-filled replication intermediates, mature cores with relaxed circular double- ...Hepatitis B virus (HBV) capsids are found in many forms: immature single-stranded RNA-filled cores, single-stranded DNA-filled replication intermediates, mature cores with relaxed circular double-stranded DNA, and empty capsids. A capsid, the protein shell of the core, is a complex of 240 copies of core protein. Mature cores are transported to the nucleus by a complex that includes both importin α and importin β (Impα and Impβ), which bind to the core protein's C-terminal domains (CTDs). Here we have investigated the interactions of HBV core protein with importins in vitro. Strikingly, empty capsids and free core protein can bind Impβ without Impα. Cryo-EM image reconstructions show that the CTDs, which are located inside the capsid, can extrude through the capsid to be bound by Impβ. Impβ density localized on the capsid exterior near the quasi-sixfold vertices, suggested a maximum of 30 Impβ per capsid. However, examination of complexes using single molecule charge-detection mass spectrometry indicate that some complexes include over 90 Impβ molecules. Cryo-EM of capsids incubated with excess Impβ shows a population of damaged particles and a population of "dark" particles with internal density, suggesting that Impβ is effectively swallowed by the capsids, which implies that the capsids transiently open and close and can be destabilized by Impβ. Though the in vitro complexes with great excess of Impβ are not biological, these results have implications for trafficking of empty capsids and free core protein; activities that affect the basis of chronic HBV infection.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionDec 1, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseAug 24, 2016-
UpdateSep 28, 2016-
Current statusSep 28, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 241
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 241
  • 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_3270.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.51 Å/pix.
x 400 pix.
= 604.8 Å
1.51 Å/pix.
x 400 pix.
= 604.8 Å
1.51 Å/pix.
x 400 pix.
= 604.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.512 Å
Density
Contour LevelBy AUTHOR: 241.0 / Movie #1: 241
Minimum - Maximum26.64142227 - 406.741790770000023
Average (Standard dev.)199.071380620000014 (±26.352891920000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-200-200-200
Dimensions400400400
Spacing400400400
CellA=B=C: 604.7999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.5121.5121.512
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z604.800604.800604.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS-200-200-200
NC/NR/NS400400400
D min/max/mean26.641406.742199.071

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Supplemental data

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Sample components

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Entire Name:11 uM Cp183 dimer in capsid form and 18.8 uM Imp-beta in amm...

EntireName: Name:11 uM Cp183 dimer in capsid form and 18.8 uM Imp-beta in ammonium formate (dark particles)
Number of components: 2

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Component #1: virus, Hepatitis B virus

VirusName: Hepatitis B virus / a.k.a: HBV / Class: VIRUS-LIKE PARTICLE / Enveloped: No / Empty: No / Isolate: STRAIN
MassTheoretical: 4.8 MDa / Experimental: 4.8 MDa
SpeciesSpecies: Hepatitis B virus (HBV) / Strain: adyw
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET11cCp183 / Strain: BL21 (DE3) cells
Source (natural)Host Species: Homo sapiens (human) / Host category: VERTEBRATES
Shell #1Name of element: Cp183 / T number (triangulation number): 4

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Component #2: protein, importin beta

ProteinName: importin betaImportin / a.k.a: Imp-beta / Recombinant expression: Yes
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: PET30a

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.39 mg/mL / Buffer solution: 0.15M NaCl, 10 mM DTT, 20 mM Tris-HCl / pH: 7.4
Support filmglow-discharged holey carbon grid (Quantifoil R2/2) or continuous carbon film coated grid (EMS)
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 98 K / Humidity: 100 % / Method: Blot for 4 seconds before plunging

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Electron microscopy imaging

ImagingMicroscope: JEOL 3200FS / Date: Jun 19, 2014 / Details: Weak beam illumination
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 80000 X (nominal) / Cs: 1.1 mm / Imaging mode: BRIGHT FIELD / Defocus: 1560 - 5460 nm / Energy filter: Omega filter / Energy window: 0-20 eV
Specimen HolderHolder: Gatan 626 / Model: GATAN LIQUID NITROGEN / Temperature: 98
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 129 / Sampling size: 15 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 3154 / Details: Data processed by auto3dem
3D reconstructionSoftware: Auto3dem / CTF correction: each particle / Resolution: 13.8 Å / Resolution method: FSC at 0.143 cut-off

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