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- PDB-6ygi: Duck hepatitis B virus capsid Mutant R124E_delta78-122 -

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Basic information

Entry
Database: PDB / ID: 6ygi
TitleDuck hepatitis B virus capsid Mutant R124E_delta78-122
ComponentsCapsid protein,Capsid protein
KeywordsVIRUS LIKE PARTICLE / duck hepatitis B core protein / extension domain / spike / slowly folding
Function / homology
Function and homology information


microtubule-dependent intracellular transport of viral material towards nucleus / T=4 icosahedral viral capsid / viral penetration into host nucleus / host cell cytoplasm / symbiont entry into host cell / structural molecule activity / DNA binding / RNA binding
Similarity search - Function
Hepatitis core antigen / Viral capsid core domain supefamily, Hepatitis B virus / Hepatitis core antigen
Similarity search - Domain/homology
Biological speciesHepatitis B virus duck/DHBV-16
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsMakbul, C. / Bottcher, B.
Funding support Germany, 3items
OrganizationGrant numberCountry
German Research Foundation (DFG)Bo1150/17-1 Germany
German Research Foundation (DFG)INST 93/903-1 FUGG Germany
German Research Foundation (DFG)Na154/9-4 Germany
CitationJournal: Elife / Year: 2020
Title: Slowly folding surface extension in the prototypic avian hepatitis B virus capsid governs stability.
Authors: Cihan Makbul / Michael Nassal / Bettina Böttcher /
Abstract: Hepatitis B virus (HBV) is an important but difficult to study human pathogen. Most basics of the hepadnaviral life-cycle were unraveled using duck HBV (DHBV) as a model although DHBV has a capsid ...Hepatitis B virus (HBV) is an important but difficult to study human pathogen. Most basics of the hepadnaviral life-cycle were unraveled using duck HBV (DHBV) as a model although DHBV has a capsid protein (CP) comprising ~260 rather than ~180 amino acids. Here we present high-resolution structures of several DHBV capsid-like particles (CLPs) determined by electron cryo-microscopy. As for HBV, DHBV CLPs consist of a dimeric α-helical frame-work with protruding spikes at the dimer interface. A fundamental new feature is a ~ 45 amino acid proline-rich extension in each monomer replacing the tip of the spikes in HBV CP. In vitro, folding of the extension takes months, implying a catalyzed process in vivo. DHBc variants lacking a folding-proficient extension produced regular CLPs in bacteria but failed to form stable nucleocapsids in hepatoma cells. We propose that the extension domain acts as a conformational switch with differential response options during viral infection.
History
DepositionMar 27, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
E: Capsid protein,Capsid protein
H: Capsid protein,Capsid protein
A: Capsid protein,Capsid protein
B: Capsid protein,Capsid protein
C: Capsid protein,Capsid protein
D: Capsid protein,Capsid protein


Theoretical massNumber of molelcules
Total (without water)152,9766
Polymers152,9766
Non-polymers00
Water0
1
E: Capsid protein,Capsid protein
H: Capsid protein,Capsid protein
A: Capsid protein,Capsid protein
B: Capsid protein,Capsid protein
C: Capsid protein,Capsid protein
D: Capsid protein,Capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)9,178,556360
Polymers9,178,556360
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
MethodUCSF CHIMERA

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Components

#1: Protein
Capsid protein,Capsid protein / / Core antigen / Core protein / HBcAg


Mass: 25495.988 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: deletion mutant with extension domain (78-122) replaced by flexible linker GGSGG additional point mutation R124E,deletion mutant with extension domain (78-122) replaced by flexible linker ...Details: deletion mutant with extension domain (78-122) replaced by flexible linker GGSGG additional point mutation R124E,deletion mutant with extension domain (78-122) replaced by flexible linker GGSGG additional point mutation R124E
Source: (gene. exp.) Hepatitis B virus duck/DHBV-16 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0C6J7

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Hepatitis B virus duck/DHBV-16 / Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 7 MDa / Experimental value: NO
Source (natural)Organism: Hepatitis B virus duck/DHBV-16
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Anas platyrhynchos
Virus shellName: duck Hepatitis B virus capsid / Diameter: 370 nm / Triangulation number (T number): 4
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
250 mMNaClSodium chloride1
31 mMMgCl21
41 mMCaCl21
55 mMDTT1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: For the vitrification, grids (400 mesh copper grids (type R 1.2/1.3. Quantifoil Micro Tools, Jena/Germany) were rendered hydrophilic by glow discharging in air at a pressure of 29 Pa for 2 ...Details: For the vitrification, grids (400 mesh copper grids (type R 1.2/1.3. Quantifoil Micro Tools, Jena/Germany) were rendered hydrophilic by glow discharging in air at a pressure of 29 Pa for 2 minutes at medium power with a Plasma Cleaner (model PDC-002. Harrick Plasma Ithaca, NY/USA). Then, 3.5 ul of DHBc solution was pipetted onto the grids and they were plunge frozen in liquid ethane with a Vitrobot mark IV (FEI-Thermo Fisher Scientific). The settings for the Vitrobot were 3s blot time, 45 s wait time, blot force 0 at a temperature of 4 C and 100 % humidity

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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 X / Calibrated defocus min: 500 nm / Calibrated defocus max: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 54 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1496
Details: movie mode, 2 images per hole, 40 fractions per movie
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFINDCTF correctiondeterminingctf
5RELIONCTF correctionapplying ctf correction
8UCSF Chimeramodel fitting
9Cootmodel fitting
11RELIONinitial Euler assignment
12RELION3final Euler assignmentauto_refinement
13RELION3classification
14RELION33D reconstruction
15PHENIXmodel refinement
Image processingDetails: Micrographs were dose weighted and motion corrected with Motioncor2. The ctf was determined with ctffind4
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 74760
Details: particles were automatically selected using 2 D class averages as template
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44498 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310192
ELECTRON MICROSCOPYf_angle_d0.53618355
ELECTRON MICROSCOPYf_dihedral_angle_d12.6064098
ELECTRON MICROSCOPYf_chiral_restr0.034760
ELECTRON MICROSCOPYf_plane_restr0.0031512

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