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- EMDB-2509: Cryo-EM structure of immature HBV core -

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Basic information

Entry
Database: EMDB / ID: EMD-2509
TitleCryo-EM structure of immature HBV core
Map datacryo-EM 3-D reconstruction of immature pgRNA-filled HBV core
Sample
  • Sample: Immature pgRNA-filled HBV Core
  • Virus: Hepatitis B virus
KeywordsHBV / hepatitis B virus / pgRNA
Biological speciesHepatitis B virus
Methodsingle particle reconstruction / cryo EM / Resolution: 14.5 Å
AuthorsWang JC-Y / Nickens DG / Lentz TB / Loeb DD / Zlotnick A
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice.
Authors: Joseph Che-Yen Wang / David G Nickens / Thomas B Lentz / Daniel D Loeb / Adam Zlotnick /
Abstract: Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse ...Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well.
History
DepositionNov 14, 2013-
Header (metadata) releaseNov 27, 2013-
Map releaseJul 30, 2014-
UpdateAug 13, 2014-
Current statusAug 13, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0776
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0776
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2509.jpg

Downloads & links

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Map

FileDownload / File: emd_2509.map.gz / Format: CCP4 / Size: 15 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM 3-D reconstruction of immature pgRNA-filled HBV core
Voxel sizeX=Y=Z: 2.94 Å
Density
Contour LevelBy AUTHOR: 0.0776 / Movie #1: 0.0776
Minimum - Maximum-0.3463417 - 0.36404872
Average (Standard dev.)0.01064173 (±0.04804695)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-79-79-79
Dimensions159159159
Spacing159159159
CellA=B=C: 467.46002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.942.942.94
M x/y/z159159159
origin x/y/z0.0000.0000.000
length x/y/z467.460467.460467.460
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-79-79-79
NC/NR/NS159159159
D min/max/mean-0.3460.3640.011

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Supplemental data

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Sample components

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Entire : Immature pgRNA-filled HBV Core

EntireName: Immature pgRNA-filled HBV Core
Components
  • Sample: Immature pgRNA-filled HBV Core
  • Virus: Hepatitis B virus

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Supramolecule #1000: Immature pgRNA-filled HBV Core

SupramoleculeName: Immature pgRNA-filled HBV Core / type: sample / ID: 1000 / Details: Sample was purified from Huh7-H1 / Number unique components: 1

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Supramolecule #1: Hepatitis B virus

SupramoleculeName: Hepatitis B virus / type: virus / ID: 1 / NCBI-ID: 10407 / Sci species name: Hepatitis B virus / Sci species strain: subtype ayw, genotype D / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: Homo sapiens (human) / Recombinant cell: Huh7-H1 / Recombinant plasmid: p1159, p1929
Virus shellShell ID: 1 / Name: Cp / Diameter: 340 Å / T number (triangulation number): 4

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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
GridDetails: 300 mesh continuous carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 4 seconds before plunging

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Electron microscopy #1

MicroscopeJEOL 3200FS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.1 mm / Nominal magnification: 40000
Specialist opticsEnergy filter - Name: Omega filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: 626 / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 97 K
Microscopy ID1
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 80,000 times magnification
DateDec 20, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 513 / Average electron dose: 20 e/Å2

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Electron microscopy #2

MicroscopeJEOL 3200FS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.1 mm / Nominal magnification: 40000
Specialist opticsEnergy filter - Name: Omega filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: 626 / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 97 K
Microscopy ID2
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 80,000 times magnification
DateDec 21, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 513 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: Each particle phase-flipping
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 14.5 Å / Resolution method: OTHER / Software - Name: AUTO3DEM / Number images used: 11727

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