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- EMDB-10316: Hepatitis B virus core protein virus-like particle displaying the... -

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Basic information

Entry
Database: EMDB / ID: EMD-10316
TitleHepatitis B virus core protein virus-like particle displaying the antigen: extra cellular adhesion domain NadA from Neisseria meningitidis.
Map dataHepatitis B virus core protein with the extracellular domain of NadA inserted at the tip of the spikes of the VLP/capsid spikes.
Sample
  • Complex: VLP fusion sequence expressed in E.coli.
    • Protein or peptide: HBcS-NadA-CFHbp
Function / homology
Function and homology information


microtubule-dependent intracellular transport of viral material towards nucleus / T=4 icosahedral viral capsid / cell outer membrane / viral penetration into host nucleus / host cell cytoplasm / membrane => GO:0016020 / symbiont entry into host cell / viral envelope / structural molecule activity / cell surface ...microtubule-dependent intracellular transport of viral material towards nucleus / T=4 icosahedral viral capsid / cell outer membrane / viral penetration into host nucleus / host cell cytoplasm / membrane => GO:0016020 / symbiont entry into host cell / viral envelope / structural molecule activity / cell surface / DNA binding / RNA binding
Similarity search - Function
: / : / Factor H binding protein, N-terminal / Factor H binding protein, C-terminal / Factor H binding protein, C-terminal / Trimeric autotransporter adhesin YadA-like, C-terminal membrane anchor domain / YadA-like membrane anchor domain / Hepatitis core antigen / Viral capsid core domain supefamily, Hepatitis B virus / Hepatitis core antigen ...: / : / Factor H binding protein, N-terminal / Factor H binding protein, C-terminal / Factor H binding protein, C-terminal / Trimeric autotransporter adhesin YadA-like, C-terminal membrane anchor domain / YadA-like membrane anchor domain / Hepatitis core antigen / Viral capsid core domain supefamily, Hepatitis B virus / Hepatitis core antigen / Outer membrane protein/outer membrane enzyme PagP, beta-barrel / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Putative adhesin/invasin / Capsid protein / Factor H-binding protein
Similarity search - Component
Biological speciesNeisseria meningitidis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsCollins RF / Roseman AM / Derrick JP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBRIC PhD, BBSRC Reference BB/K02034X/1 United Kingdom
CitationJournal: Vaccine / Year: 2020
Title: An assessment of the use of Hepatitis B Virus core protein virus-like particles to display heterologous antigens from Neisseria meningitidis.
Authors: Sebastian Aston-Deaville / Emil Carlsson / Muhammad Saleem / Angela Thistlethwaite / Hannah Chan / Sunil Maharjan / Alessandra Facchetti / Ian M Feavers / C Alistair Siebert / Richard F ...Authors: Sebastian Aston-Deaville / Emil Carlsson / Muhammad Saleem / Angela Thistlethwaite / Hannah Chan / Sunil Maharjan / Alessandra Facchetti / Ian M Feavers / C Alistair Siebert / Richard F Collins / Alan Roseman / Jeremy P Derrick /
Abstract: Neisseria meningitidis is the causative agent of meningococcal meningitis and sepsis and remains a significant public health problem in many countries. Efforts to develop a comprehensive vaccine ...Neisseria meningitidis is the causative agent of meningococcal meningitis and sepsis and remains a significant public health problem in many countries. Efforts to develop a comprehensive vaccine against serogroup B meningococci have focused on the use of surface-exposed outer membrane proteins. Here we report the use of virus-like particles derived from the core protein of Hepatitis B Virus, HBc, to incorporate antigen domains derived from Factor H binding protein (FHbp) and the adhesin NadA. The extracellular domain of NadA was inserted into the major immunodominant region of HBc, and the C-terminal domain of FHbp at the C-terminus (CFHbp), creating a single polypeptide chain 3.7-fold larger than native HBc. Remarkably, cryoelectron microscopy revealed that the construct formed assemblies that were able to incorporate both antigens with minimal structural changes to native HBc. Electron density was weak for NadA and absent for CFHbp, partly attributable to domain flexibility. Following immunization of mice, three HBc fusions (CFHbp or NadA alone, NadA + CFHbp) were able to induce production of IgG1, IgG2a and IgG2b antibodies reactive against their respective antigens at dilutions in excess of 1:18,000. However, only HBc fusions containing NadA elicited the production of antibodies with serum bactericidal activity. It is hypothesized that this improved immune response is attributable to the adoption of a more native-like folding of crucial conformational epitopes of NadA within the chimeric VLP. This work demonstrates that HBc can incorporate insertions of large antigen domains but that maintenance of their three-dimensional structure is likely to be critical in obtaining a protective response.
History
DepositionSep 18, 2019-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tik
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tik
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10316.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHepatitis B virus core protein with the extracellular domain of NadA inserted at the tip of the spikes of the VLP/capsid spikes.
Voxel sizeX=Y=Z: 1.39 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-1.1796018 - 4.9631643
Average (Standard dev.)0.011435016 (±0.23314595)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 711.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z711.680711.680711.680
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-1.1804.9630.011

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Supplemental data

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Additional map: Unsharpened map

Fileemd_10316_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Mask for FSC calculation

Fileemd_10316_additional_2.map
AnnotationMask for FSC calculation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map A.

Fileemd_10316_half_map_1.map
AnnotationHalf map A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map B.

Fileemd_10316_half_map_2.map
AnnotationHalf map B.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : VLP fusion sequence expressed in E.coli.

EntireName: VLP fusion sequence expressed in E.coli.
Components
  • Complex: VLP fusion sequence expressed in E.coli.
    • Protein or peptide: HBcS-NadA-CFHbp

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Supramolecule #1: VLP fusion sequence expressed in E.coli.

SupramoleculeName: VLP fusion sequence expressed in E.coli. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: HBcS-NadA-CFHbp Fusion protein with linker regions. HBc - Ordered core of VLP: from HBV core protein (NCBI taxonomy ID 10407). NadA - Partially ordered surface displayed domain: ...Details: HBcS-NadA-CFHbp Fusion protein with linker regions. HBc - Ordered core of VLP: from HBV core protein (NCBI taxonomy ID 10407). NadA - Partially ordered surface displayed domain: extracellular domain of NadA from Neisseria meningitidis (NCBI taxonomy ID 487). CFHbp - Unresolved/disordered component : C-terminal domain of factor H binding protein from Neisseria meningitidis (NCBI taxonomy ID 487).
Source (natural)Organism: Neisseria meningitidis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET-17b
Molecular weightTheoretical: 14.9 MDa

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Macromolecule #1: HBcS-NadA-CFHbp

MacromoleculeName: HBcS-NadA-CFHbp / type: protein_or_peptide / ID: 1 / Details: expressed fusion chimera protein / Enantiomer: LEVO
SequenceString: MDIDPYKEFG ATVELLSFLP SDFFPSVRDL LDTASALYRE ALESPEHCSP HHTALRQAI LCWGELMTLA TWVGNNLEDG GGGSGGGGSA TSDDDVKKAA TVAIVAAYNN GQEINGFKAG ETIYDIGED GTITQKDATA ADVEADDFKG LGLKKVVTNL TKTVNENKQN ...String:
MDIDPYKEFG ATVELLSFLP SDFFPSVRDL LDTASALYRE ALESPEHCSP HHTALRQAI LCWGELMTLA TWVGNNLEDG GGGSGGGGSA TSDDDVKKAA TVAIVAAYNN GQEINGFKAG ETIYDIGED GTITQKDATA ADVEADDFKG LGLKKVVTNL TKTVNENKQN VDAKVKAAES E IEKLTTKL ADTDAALADT DAALDETTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA DT VDKHAEA FNDIADSLDE TNTKADEAVK TANEAKQTAE ETKQNVDAKV KAAETAAGKA EAA AGTANT AADKAEAVAA KVTDIKADIA TNKADIAKNS ARIDSLDKNV ANLRKETRQG LAEQ AALSG LFQPYNVGEF GGGGSGGGGS RDLVVNYVNT NMGLKIRQLL WFHISCLTFG RETVL EYLV SFGVWIRTPP AYRPPNAPIL STLPETTVVG SGGGTHTSFD KLPEGGRATY RGTAFG SDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAE KG SYSLGIFGGK AQEVAGSAEV KTVNGIRHIG LAAKQKLGGG WSHPQFEK

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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

Concentration1 mg/mL
BufferpH: 8 / Details: PBS
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: 3 ul applied for 30s at room temperature, then 4-5 s blot..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.84 µm / Calibrated defocus min: 0.424 µm / Calibrated magnification: 100719 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.0025 µm / Nominal defocus min: 0.001 µm / Nominal magnification: 104167
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Digitization - Frames/image: 3-36 / Number grids imaged: 1 / Number real images: 2367 / Average exposure time: 4.0 sec. / Average electron dose: 79.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 10119
Details: Initially ~44,000 particles were initially picked and extracted in EMAN. Approx 12,000 were shell fragments or incomplete. Others were intact but lower resolution, so damaged or affected by ...Details: Initially ~44,000 particles were initially picked and extracted in EMAN. Approx 12,000 were shell fragments or incomplete. Others were intact but lower resolution, so damaged or affected by charging, or other issue. After filtering/cleaning by EMAN2 2D classification 10119 particles were taken forwards for initial 3D reconstruction in EMAN2. This set of 10119 was then 2D cleaned by CryoSPARC to give 9145 particles.
CTF correctionSoftware - Name: EMAN2 (ver. 2.01) / Software - details: then CTF applied by CryoSPARC
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v0.65)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. v0.65) / Details: model #0 94.2 % mode l#1 4.6 % model #2 1.2%
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v0.65)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v0.65) / Number images used: 8598

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