|Entry||Database: EMDB / ID: EMD-3270|
|Title||Importin-beta can bind Hepatitis B Virus core protein and empty core-like particles and induce structural changes|
|Sample||Name:11 uM Cp183 dimer in capsid form and 18.8 uM Imp-beta in ammonium formate (dark particles):|
virus / importin betaImportin
|Keywords||HBV / Cp183 / Importin-beta|
|Biological species||Hepatitis B virus (HBV) / Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 13.8 Å|
|Authors||Chen C / Wang JC-Y / Pierson EE / Kiefer DZ / Delaleau M / Gallucci L / Cazenave C / Kann M / Jarrold MF / Zlotnick A|
|Citation||Journal: 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 Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_3270.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.512 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Name:11 uM Cp183 dimer in capsid form and 18.8 uM Imp-beta in amm...
|Entire||Name: Name:11 uM Cp183 dimer in capsid form and 18.8 uM Imp-beta in ammonium formate (dark particles)|
Number of components: 2
-Component #1: virus, Hepatitis B virus
|Virus||Name: Hepatitis B virus / a.k.a: HBV / Class: VIRUS-LIKE PARTICLE / Enveloped: No / Empty: No / Isolate: STRAIN|
|Mass||Theoretical: 4.8 MDa / Experimental: 4.8 MDa|
|Species||Species: 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 #1||Name of element: Cp183 / T number (triangulation number): 4|
-Component #2: protein, importin beta
|Protein||Name: importin betaImportin / a.k.a: Imp-beta / Recombinant expression: Yes|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Escherichia coli (E. coli) / Vector: PET30a|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.39 mg/mL / Buffer solution: 0.15M NaCl, 10 mM DTT, 20 mM Tris-HCl / pH: 7.4|
|Support film||glow-discharged holey carbon grid (Quantifoil R2/2) or continuous carbon film coated grid (EMS)|
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 98 K / Humidity: 100 % / Method: Blot for 4 seconds before plunging|
-Electron microscopy imaging
|Imaging||Microscope: JEOL 3200FS / Date: Jun 19, 2014 / Details: Weak beam illumination|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 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 Holder||Holder: Gatan 626 / Model: GATAN LIQUID NITROGEN / Temperature: 98|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Image acquisition||Number of digital images: 129 / Sampling size: 15 µm|
|Processing||Method: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 3154 / Details: Data processed by auto3dem|
|3D reconstruction||Software: Auto3dem / CTF correction: each particle / Resolution: 13.8 Å / Resolution method: FSC at 0.143 cut-off|
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