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- EMDB-10802: The Mutant Duck Hepatitis B core protein R124E has a disordered e... -

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Basic information

Entry
Database: EMDB / ID: EMD-10802
TitleThe Mutant Duck Hepatitis B core protein R124E has a disordered extension domain
Map data
Sample
  • Virus: Hepatitis B virus duck/DHBV-16
    • Protein or peptide: Duck Hepatitis B Virus Capsid Protein Mutant R124E
Biological speciesHepatitis B virus duck/DHBV-16
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsMakbul C / Bottcher B
Funding support Germany, 3 items
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, 2020-
Header (metadata) releaseAug 26, 2020-
Map releaseAug 26, 2020-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10802.png

Downloads & links

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Map

FileDownload / File: emd_10802.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0635 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.01575307 - 0.03598799
Average (Standard dev.)0.0008236803 (±0.0033130252)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 425.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06351.06351.0635
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z425.400425.400425.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0160.0360.001

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Supplemental data

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Mask #1

Fileemd_10802_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10802_half_map_1.map
Projections & Slices
AxesZYX

Projections

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10802_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : Hepatitis B virus duck/DHBV-16

EntireName: Hepatitis B virus duck/DHBV-16
Components
  • Virus: Hepatitis B virus duck/DHBV-16
    • Protein or peptide: Duck Hepatitis B Virus Capsid Protein Mutant R124E

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Supramolecule #1: Hepatitis B virus duck/DHBV-16

SupramoleculeName: Hepatitis B virus duck/DHBV-16 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 489543 / Sci species name: Hepatitis B virus duck/DHBV-16 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Anas platyrhynchos (mallard)
Host systemOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 7.2 MDa
Virus shellShell ID: 1 / Name: duck Hepatitis B Virus capsid / Diameter: 370.0 Å / T number (triangulation number): 4

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Macromolecule #1: Duck Hepatitis B Virus Capsid Protein Mutant R124E

MacromoleculeName: Duck Hepatitis B Virus Capsid Protein Mutant R124E / type: protein_or_peptide / ID: 1
Details: Mutant with Arg 124 replaced by Glutamate -> charge reversal
Enantiomer: LEVO
Source (natural)Organism: Hepatitis B virus duck/DHBV-16
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MDINASRALA NVYDLPDDFF PKIDDLVRDA KDALEPYWKS DSIKKHVLIA THFVDLIEDF WQTTQGMHE IAESLRAVIP PTTTPVPPGY LIQHEEAEEI PLGDLFKHQE ERIVSFQPDY P ITAEIHAH LKAYAKINEE SLDRARRLLW WHYNCLLWGE AQVTNYISRL ...String:
MDINASRALA NVYDLPDDFF PKIDDLVRDA KDALEPYWKS DSIKKHVLIA THFVDLIEDF WQTTQGMHE IAESLRAVIP PTTTPVPPGY LIQHEEAEEI PLGDLFKHQE ERIVSFQPDY P ITAEIHAH LKAYAKINEE SLDRARRLLW WHYNCLLWGE AQVTNYISRL RTWLSTPEKY RG RDAPTIE AITRPIQVAQ GGRKTTTGTR KPRGLEPRRR KVKTTVVYGR RRSKSRERRA PTP QRAGSP LPRSSSSHHR SPSPRK

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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 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris
50.0 mMNaClSodium chloride
1.0 mMMgCl2
1.0 mMCaCl2
5.0 mMDTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 75000
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 / Number grids imaged: 1 / Number real images: 1226 / Average exposure time: 2.3 sec. / Average electron dose: 40.0 e/Å2
Details: movie mode, 2 images per hole, 20 fractions per movie
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 35820
Details: particles were automatically selected using 2 D class averages as template
CTF correctionSoftware:
Namedetails
CTFFINDdeterminingctf
RELIONapplying ctf correction
Startup modelType of model: OTHER / Details: wtDHBC map low pass filtered to 25 A
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3) / Software - details: auto_refinement
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 23384
DetailsMicrographs were dose weighted and motion corrected with Motioncor2. The ctf was determined with ctffind4
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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