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- PDB-6hu7: phosphorylated F97L Hepatitis B core protein capsid -

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Basic information

Entry
Database: PDB / ID: 6hu7
Titlephosphorylated F97L Hepatitis B core protein capsid
ComponentsCapsid proteinCapsid
KeywordsVIRUS LIKE PARTICLE / Hapatitis B core protein Hbc Hbcag / VIRUS LIKE PARTICLE premature envelopment mutant phosphorylated F97L
Function / homologyHepatitis core antigen / Hepatitis core protein, putative zinc finger / Hepatitis core antigen / Hepatitis B virus, capsid N-terminal / Viral capsid core domain supefamily, Hepatitis B virus / microtubule-dependent intracellular transport of viral material towards nucleus / T=4 icosahedral viral capsid / evasion or tolerance by virus of host immune response / viral penetration into host nucleus / viral entry into host cell ...Hepatitis core antigen / Hepatitis core protein, putative zinc finger / Hepatitis core antigen / Hepatitis B virus, capsid N-terminal / Viral capsid core domain supefamily, Hepatitis B virus / microtubule-dependent intracellular transport of viral material towards nucleus / T=4 icosahedral viral capsid / evasion or tolerance by virus of host immune response / viral penetration into host nucleus / viral entry into host cell / host cell cytoplasm / pathogenesis / structural molecule activity / RNA binding / DNA binding / extracellular region / Capsid protein
Function and homology information
Specimen sourceHepatitis B virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 2.8 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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Structure viewerMolecule:
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Assembly

Deposited unit
B: Capsid protein
A: Capsid protein
C: Capsid protein
D: Capsid protein
F: Capsid protein
G: Capsid protein
H: Capsid protein


Theoretical massNumber of molelcules
Total (without water)147,7857
Polyers147,7857
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)11130
ΔGint (kcal/M)-96
Surface area (Å2)30410
MethodPISA

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Components

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


Mass: 21112.199 Da / Num. of mol.: 7 / Mutation: F97L / Source: (gene. exp.) Hepatitis B virus / Production host: Escherichia coli (E. coli) / References: UniProt: D0EYZ6

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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 / Strain: ayw
Source (recombinant)Organism: Escherichia coli (E. coli)
Details of virusEmpty: YES / Enveloped: NO / Virus isolate: STRAIN / Virus type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Virus shellName: F97L Hbc phos / 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: 278 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: 600 nm / Calibrated defocus max: 5000 nm / Cs: 0 mm / C2 aperture diameter: 70 microns / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Number of grids imaged: 1 / Number of real images: 2341
EM imaging opticsSph aberration corrector: cs-corrected
Image scansMovie frames/image: 28 / Used frames/image: 1-28

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION2.1particle selection
2EPUimage acquisition3 movies per hole
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: 94887
SymmetryPoint symmetry: I
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 31635 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingOverall b value: 102 / Ref protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 6HU4
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0085030
ELECTRON MICROSCOPYf_angle_d0.7656884
ELECTRON MICROSCOPYf_dihedral_angle_d10.4774550
ELECTRON MICROSCOPYf_chiral_restr0.050790
ELECTRON MICROSCOPYf_plane_restr0.007872

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