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- EMDB-3267: Importin-beta can bind Hepatitis B Virus core protein and empty c... -

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Entry
Database: EMDB / ID: EMD-3267
TitleImportin-beta can bind Hepatitis B Virus core protein and empty core-like particles and induce structural changes
Map dataCryo-EM reconstruction of phosphorylated Cp183 and Imp-beta in NaCl
Sample
  • Sample: Phosphorylated 7.9 uM Cp183 dimer in capsid form and 5.3 uM Imp-beta in NaCl
  • Virus: Hepatitis B virus
  • Protein or peptide: importin beta
KeywordsHBV / Cp183 / Importin-beta
Biological speciesHomo sapiens (human) / Hepatitis B virus
Methodsingle particle reconstruction / cryo EM / Resolution: 10.9 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.
History
DepositionDec 1, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseAug 24, 2016-
UpdateAug 24, 2016-
Current statusAug 24, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.33
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.33
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3267.png

Downloads & links

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Map

FileDownload / File: emd_3267.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of phosphorylated Cp183 and Imp-beta in NaCl
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.33 / Movie #1: 0.33
Minimum - Maximum-0.59045798 - 1.31045985
Average (Standard dev.)0.02011116 (±0.16657774)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-165-165-165
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
MAP C/R/S123
start NC/NR/NS-165-165-165
NC/NR/NS400400400
D min/max/mean-0.5901.3100.020

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Supplemental data

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Sample components

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Entire : Phosphorylated 7.9 uM Cp183 dimer in capsid form and 5.3 uM Imp-b...

EntireName: Phosphorylated 7.9 uM Cp183 dimer in capsid form and 5.3 uM Imp-beta in NaCl
Components
  • Sample: Phosphorylated 7.9 uM Cp183 dimer in capsid form and 5.3 uM Imp-beta in NaCl
  • Virus: Hepatitis B virus
  • Protein or peptide: importin beta

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Supramolecule #1000: Phosphorylated 7.9 uM Cp183 dimer in capsid form and 5.3 uM Imp-b...

SupramoleculeName: Phosphorylated 7.9 uM Cp183 dimer in capsid form and 5.3 uM Imp-beta in NaCl
type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: Hepatitis B virus

SupramoleculeName: Hepatitis B virus / type: virus / ID: 1 / Name.synonym: HBV / NCBI-ID: 10407 / Sci species name: Hepatitis B virus / Sci species strain: adyw / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes / Syn species name: HBV
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) cells / Recombinant plasmid: pET11cCp183
Molecular weightExperimental: 4.8 MDa / Theoretical: 4.8 MDa
Virus shellShell ID: 1 / Name: Cp183 / T number (triangulation number): 4

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Macromolecule #1: importin beta

MacromoleculeName: importin beta / type: protein_or_peptide / ID: 1 / Name.synonym: Imp-beta / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: PET30a

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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

Concentration0.28 mg/mL
BufferpH: 7.4 / Details: 0.15M NaCl, 10 mM DTT, 20 mM Tris-HCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 98 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 4 seconds before plunging

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Electron microscopy

MicroscopeJEOL 3200FS
TemperatureAverage: 98 K
Specialist opticsEnergy filter - Name: Omega filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DetailsWeak beam illumination
DateJul 30, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 81 / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.1 mm / Nominal defocus max: 4.36 µm / Nominal defocus min: 1.07 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsData processed by auto3dem
CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 10.9 Å / Resolution method: OTHER / Software - Name: Auto3dem / Number images used: 5760

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