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- EMDB-11701: HBV pgRNA T=4 NCP non-icosahedral symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-11701
TitleHBV pgRNA T=4 NCP non-icosahedral symmetry
Map dataHBV T=4 NCP asymmetry
Sample
  • Virus: Hepatitis B virus
    • Protein or peptide: Hepatitis B virus nucleocapsid protein
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 / host cell cytoplasm / symbiont entry into host cell / virus-mediated perturbation of host defense response / structural molecule activity / DNA binding / RNA binding / extracellular region
Similarity search - Function
Hepatitis B virus, capsid N-terminal / Hepatitis core protein, putative zinc finger / Hepatitis core antigen / Viral capsid core domain supefamily, Hepatitis B virus / Hepatitis core antigen
Similarity search - Domain/homology
Biological speciesHepatitis B virus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsPatel N / Clark S / Weis EU / Mata CP / Bohon J / Farquhar E / Ranson NA / Twarock R / Stockley PG
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MRF-044-0002-RG-PATEL; MR/N021517/1 United Kingdom
Wellcome TrustJoint Investigator Award Nos. 110145 & 110146; 089311/Z/09/Z; 090932/Z/09/Z & 106692 United Kingdom
CitationJournal: Commun Biol / Year: 2021
Title: In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus.
Authors: Nikesh Patel / Sam Clark / Eva U Weiß / Carlos P Mata / Jen Bohon / Erik R Farquhar / Daniel P Maskell / Neil A Ranson / Reidun Twarock / Peter G Stockley /
Abstract: The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core ...The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.
History
DepositionSep 7, 2020-
Header (metadata) releaseOct 27, 2021-
Map releaseOct 27, 2021-
UpdateMay 11, 2022-
Current statusMay 11, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11701.png

Downloads & links

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Map

FileDownload / File: emd_11701.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHBV T=4 NCP asymmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 380 pix.
= 404.7 Å		1.07 Å/pix.
x 380 pix.
= 404.7 Å		1.07 Å/pix.
x 380 pix.
= 404.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.065 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.0735818 - 0.13853215
Average (Standard dev.)0.0010886053 (±0.01061252)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-190-190-190
Dimensions380380380
Spacing380380380
CellA=B=C: 404.7 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0651.0651.065
M x/y/z380380380
origin x/y/z0.0000.0000.000
length x/y/z404.700404.700404.700
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-190-190-190
NC/NR/NS380380380
D min/max/mean-0.0740.1390.001

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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 virus nucleocapsid protein

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Supramolecule #1: Hepatitis B virus

SupramoleculeName: Hepatitis B virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 10407 / Sci species name: Hepatitis B virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Host systemOrganism: Escherichia coli (E. coli)
Virus shellShell ID: 1 / Diameter: 342.0 Å / T number (triangulation number): 4

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Macromolecule #1: Hepatitis B virus nucleocapsid protein

MacromoleculeName: Hepatitis B virus nucleocapsid protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Hepatitis B virus
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDIDPYKEFG ASVELLSFLP SDFFPSIRDL LDTASALYRE ALES PEHCS PHHTALRQAI LCWGELMNLA TWVGSNLEDP ASRELVVSYV NVNMGLKIRQ LLW FHISCL TFGRETVLEY LVSFGVWIRT PPAYRPPNAP ILSTLPETTV VRRRGRSPRR RT PSPRRRR SQSPRRRRSQ SRESQC

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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

BufferpH: 7.5
GridModel: Quantifoil / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 2658 / Average exposure time: 1.5 sec. / Average electron dose: 53.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 75000
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

Particle selectionNumber selected: 150667
CTF correctionSoftware - Name: Gctf
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 104337
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)

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