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- PDB-6htx: WT Hepatitis B core protein capsid -

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Basic information

Entry
Database: PDB / ID: 6htx
TitleWT Hepatitis B core protein capsid
ComponentsCapsid proteinCapsid
KeywordsVIRUS LIKE PARTICLE / Hapatitis B core protein Hbc Hbcag
Function / homologyViral capsid core domain supefamily, Hepatitis B virus / Hepatitis core antigen / Hepatitis core antigen / microtubule-dependent intracellular transport of viral material towards nucleus / T=4 icosahedral viral capsid / viral penetration into host nucleus / viral entry into host cell / host cell cytoplasm / pathogenesis / structural molecule activity ...Viral capsid core domain supefamily, Hepatitis B virus / Hepatitis core antigen / Hepatitis core antigen / microtubule-dependent intracellular transport of viral material towards nucleus / T=4 icosahedral viral capsid / viral penetration into host nucleus / viral entry into host cell / host cell cytoplasm / pathogenesis / structural molecule activity / RNA binding / DNA binding / Capsid protein
Function and homology information
Specimen sourceHepatitis B virus ayw/France/Tiollais/1979
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 2.66 Å resolution
AuthorsBottcher, B. / Nassal, M.
CitationJournal: J. Mol. Biol. / Year: 2018
Title: Structure of Mutant Hepatitis B Core Protein Capsids with Premature Secretion Phenotype.
Authors: Bettina Böttcher / Michael Nassal
Abstract: Hepatitis B virus is a major human pathogen that consists of a viral genome surrounded by an icosahedrally ordered core protein and a polymorphic, lipidic envelope that is densely packed with surface ...Hepatitis B virus is a major human pathogen that consists of a viral genome surrounded by an icosahedrally ordered core protein and a polymorphic, lipidic envelope that is densely packed with surface proteins. A point mutation in the core protein in which a phenylalanine at position 97 is exchanged for a smaller leucine leads to premature envelopment of the capsid before the genome maturation is fully completed. We have used electron cryo-microscopy and image processing to investigate how the point mutation affects the structure of the capsid at 2.6- to 2.8 Å-resolution. We found that in the mutant the smaller side chain at position 97 is displaced, increasing the size of an adjacent pocket in the center of the spikes of the capsid. In the mutant, this pocket is filled with an unknown density. Phosphorylation of serine residues in the unresolved C-terminal domain of the mutant leaves the structure of the ordered capsid largely unchanged. However, we were able to resolve several previously unresolved residues downstream of proline 144 that precede the phosphorylation-sites. These residues pack against the neighboring subunits and increase the inter-dimer contact suggesting that the C-termini play an important role in capsid stabilization and provide a much larger interaction interface than previously observed.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 5, 2018 / Release: Dec 26, 2018

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-0271
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Capsid protein
A: Capsid protein
C: Capsid protein
D: Capsid protein


Theoretical massNumber of molelcules
Total (without water)84,5854
Polyers84,5854
Non-polymers00
Water0
1
B: Capsid protein
A: Capsid protein
C: Capsid protein
D: Capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)5,075,092240
Polyers5,075,092240
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
B: Capsid protein
A: Capsid protein
C: Capsid protein
D: Capsid protein
x 5


  • icosahedral pentamer
  • 423 kDa, 20 polymers
Theoretical massNumber of molelcules
Total (without water)422,92420
Polyers422,92420
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
B: Capsid protein
A: Capsid protein
C: Capsid protein
D: Capsid protein
x 6


  • icosahedral 23 hexamer
  • 508 kDa, 24 polymers
Theoretical massNumber of molelcules
Total (without water)507,50924
Polyers507,50924
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1

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Components

#1: Protein/peptide
Capsid protein / Capsid / Core antigen / Core protein / HBcAg / p21.5


Mass: 21146.217 Da / Num. of mol.: 4
Source: (gene. exp.) Hepatitis B virus ayw/France/Tiollais/1979
Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): CodonPlus / References: UniProt: P03146

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Hepatitis B virus / Type: VIRUS / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 4.8 MDa / Experimental value: NO
Source (natural)Organism: Hepatitis B virus ayw/France/Tiollais/1979
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Details of virusEmpty: NO / Enveloped: NO / Virus isolate: STRAIN / Virus type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Hepatitis B virus / Strain: ayw
Virus shellName: WT Hbc / Diameter: 340 nm / Triangulation number (T number): 4
Buffer solutionDetails: 25 mM Tris pH 7.7, 150 mM NaCl / pH: 7.7
Buffer component
IDConc.NameFormulaBuffer ID
125 mMtris1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 5 mg/ml
Details: sample is purified from e.coli via sucrose density gradient. sucrose is removed via buffer exchange on a concentrator
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 kelvins
Details: Samples were vitrified with an FEI Vitrobot IV, which was operated at 100% humidity at 4C. 2.5-3.5 ul of sample were applied to grids (Quantifoil UltrAuFoil R1.3/1.2 300 mesh gold grids ) that were glow discharged in air for 1-2 min. The sample was incubated for 30 s before blotting for 3 s with a blot force between 0 and 5.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 / Calibrated defocus min: 360 nm / Calibrated defocus max: 1680 nm / Cs: 2.7 mm / C2 aperture diameter: 70 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 32 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 5400
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4RELIONCTF correctionctffind 4
7Cootmodel fitting
8UCSF Chimeramodel fitting
9PHENIXmodel fitting
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
13RELION2.1classification
14RELION2.13D reconstruction
15PHENIXmodel refinement
16Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: template based auto picking / Number of particles selected: 227949
SymmetryPoint symmetry: I
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 104105 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 1QGT
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0094888
ELECTRON MICROSCOPYf_angle_d0.8776695
ELECTRON MICROSCOPYf_dihedral_angle_d9.8804163
ELECTRON MICROSCOPYf_chiral_restr0.055761
ELECTRON MICROSCOPYf_plane_restr0.008848

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