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- EMDB-3714: Asymmetric RNA feature in T=4 HBV virus-like particle. -

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Basic information

Entry
Database: EMDB / ID: EMD-3714
TitleAsymmetric RNA feature in T=4 HBV virus-like particle.
Map dataHepatitis B VLP assembled around a 47 nucleotide ssRNA.
Sample
  • Virus: Hepatitis B virus
    • Protein or peptide: Hepatitis B core antigen
Biological speciesHepatitis B virus
Methodsingle particle reconstruction / cryo EM / Resolution: 11.4 Å
AuthorsPatel N / White SJ / Thompson RF / Bingham R / Weiss EU / Maskell DP / Zlotnick A / Dykeman E / Tuma R / Twarock R ...Patel N / White SJ / Thompson RF / Bingham R / Weiss EU / Maskell DP / Zlotnick A / Dykeman E / Tuma R / Twarock R / Ranson NA / Stockley PG
CitationJournal: Nat Microbiol / Year: 2017
Title: HBV RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promote nucleocapsid assembly.
Authors: Nikesh Patel / Simon J White / Rebecca F Thompson / Richard Bingham / Eva U Weiß / Daniel P Maskell / Adam Zlotnick / Eric Dykeman / Roman Tuma / Reidun Twarock / Neil A Ranson / Peter G Stockley /
Abstract: Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions ...Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and the hepatitis B core protein that play roles in defining the nucleocapsid assembly pathway. Using RNA SELEX and bioinformatics, we identified multiple regions in the pre-genomic RNA with high affinity for core protein dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-electron microscopy reconstruction of the T = 4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main core protein shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of core protein. These core protein-RNA contacts may play one or more roles in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for nucleocapsid assembly.
History
DepositionMay 10, 2017-
Header (metadata) releaseJun 14, 2017-
Map releaseJun 28, 2017-
UpdateAug 23, 2017-
Current statusAug 23, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3714.png

Downloads & links

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Map

FileDownload / File: emd_3714.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHepatitis B VLP assembled around a 47 nucleotide ssRNA.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 384 pix.
= 518.4 Å		1.35 Å/pix.
x 384 pix.
= 518.4 Å		1.35 Å/pix.
x 384 pix.
= 518.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.034470826 - 0.089457996
Average (Standard dev.)0.001801797 (±0.011080689)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 518.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z518.400518.400518.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0340.0890.002

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Supplemental data

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Sample components

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Entire : Hepatitis B virus

EntireName: Hepatitis B virus
Components
  • Virus: Hepatitis B virus
    • Protein or peptide: Hepatitis B core antigen

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Supramolecule #1: Hepatitis B virus

SupramoleculeName: Hepatitis B virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Is adw strain but with some mutations from deposited sequence.
NCBI-ID: 10407 / Sci species name: Hepatitis B virus / Sci species strain: adw / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host systemOrganism: Escherichia coli (E. coli)
Virus shellShell ID: 1 / T number (triangulation number): 4

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Macromolecule #1: Hepatitis B core antigen

MacromoleculeName: Hepatitis B core antigen / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Hepatitis B virus / Strain: adw
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDIDPYKEFG ATVELLSFL P SDFFPSVR DL LDTASAL YRE ALESPE HCSP HHTAL RQAIL CWGE LMTLAT WVG NNLEDPA SR DLVVNYVN T NMGLKIRQL LWFHISCLTF GRETVLEYL V SFGVWIRT PP AYRPPNA PIL STLPET TVVR RRDRG ...String:
MDIDPYKEFG ATVELLSFL P SDFFPSVR DL LDTASAL YRE ALESPE HCSP HHTAL RQAIL CWGE LMTLAT WVG NNLEDPA SR DLVVNYVN T NMGLKIRQL LWFHISCLTF GRETVLEYL V SFGVWIRT PP AYRPPNA PIL STLPET TVVR RRDRG RSPRR RTPS PRRRRS QSP RRRRSQS RE SQC

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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

Concentration3.2 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 2397 / Average exposure time: 2.5 sec. / Average electron dose: 67.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: INSILICO MODEL
In silico model: Sphere of approximate diameter to T=4 Hepatitis B particle.
Details: Made in Spider.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 11.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 10851
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
FSC plot (resolution estimation)

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