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- EMDB-3015: Using recent advances in single-particle electron cryomicroscopy ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3015
TitleUsing recent advances in single-particle electron cryomicroscopy structure determination for sub-tomogram averaging
Map datasub-tomogram averaging reconstruction of the hepatitis B capsid.
Sample
  • Sample: hepatitis B capsid
  • Protein or peptide: hepatitis B capsid
Keywordssub-tomogram averaging / RELION / maximum-likelihood / ultrastable gold substrates / hepatitis B capsid
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 cytoplasm / symbiont entry into host cell / viral envelope / structural molecule activity / DNA binding / RNA binding
Similarity search - Function
Hepatitis core antigen / Viral capsid core domain supefamily, Hepatitis B virus / Hepatitis core antigen
Similarity search - Domain/homology
Biological speciesHepatitis B virus
Methodsubtomogram averaging / cryo EM / Resolution: 8.1 Å
AuthorsBharat TA / Russo CJ / Lowe J / Passmore LA / Scheres SHW
CitationJournal: Structure / Year: 2015
Title: Advances in Single-Particle Electron Cryomicroscopy Structure Determination applied to Sub-tomogram Averaging.
Authors: Tanmay A M Bharat / Christopher J Russo / Jan Löwe / Lori A Passmore / Sjors H W Scheres /
Abstract: Recent innovations in specimen preparation, data collection, and image processing have led to improved structure determination using single-particle electron cryomicroscopy (cryo-EM). Here we explore ...Recent innovations in specimen preparation, data collection, and image processing have led to improved structure determination using single-particle electron cryomicroscopy (cryo-EM). Here we explore some of these advances to improve structures determined using electron cryotomography (cryo-ET) and sub-tomogram averaging. We implement a new three-dimensional model for the contrast transfer function, and use this in a regularized likelihood optimization algorithm as implemented in the RELION program. Using direct electron detector data, we apply both single-particle analysis and sub-tomogram averaging to analyze radiation-induced movements of the specimen. As in single-particle cryo-EM, we find that significant sample movements occur during tomographic data acquisition, and that these movements are substantially reduced through the use of ultrastable gold substrates. We obtain a sub-nanometer resolution structure of the hepatitis B capsid, and show that reducing radiation-induced specimen movement may be central to attempts at further improving tomogram quality and resolution.
History
DepositionMay 14, 2015-
Header (metadata) releaseMay 27, 2015-
Map releaseAug 26, 2015-
UpdateSep 16, 2015-
Current statusSep 16, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMDB-3015-im...

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Map

FileDownload / File: emd_3015.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsub-tomogram averaging reconstruction of the hepatitis B capsid.
Voxel sizeX=Y=Z: 2.17 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.25
Minimum - Maximum-0.08122995 - 0.4787958
Average (Standard dev.)0.01218119 (±0.05422338)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 520.80005 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.172.172.17
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z520.800520.800520.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-21-120
NX/NY/NZ432573
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0810.4790.012

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Supplemental data

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Sample components

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Entire : hepatitis B capsid

EntireName: hepatitis B capsid
Components
  • Sample: hepatitis B capsid
  • Protein or peptide: hepatitis B capsid

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Supramolecule #1000: hepatitis B capsid

SupramoleculeName: hepatitis B capsid / type: sample / ID: 1000 / Oligomeric state: icosahedral / Number unique components: 1
Molecular weightTheoretical: 3.8 MDa

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Macromolecule #1: hepatitis B capsid

MacromoleculeName: hepatitis B capsid / type: protein_or_peptide / ID: 1 / Details: 10 nm gold particles were mixed with the sample. / Number of copies: 240 / Oligomeric state: icosahedral / Recombinant expression: Yes
Source (natural)Organism: Hepatitis B virus
Molecular weightTheoretical: 3.8 MDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceUniProtKB: Capsid protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

GridDetails: 300 mesh ultrastable gold substrates with 1.2 micron holes (Russo and Passmore, Science 2014)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.6 µm / Nominal defocus min: 3.3 µm
Specialist opticsEnergy filter - Name: Gatan Quantum Energy Filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
DetailsData was collected using SerialEM.
DateAug 28, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 60 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: RELION 3D CTF Model
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: OTHER / Software - Name: IMOD, Tomo3D, RELION / Details: gold-standard FSC measurement was used. / Number subtomograms used: 1145
DetailsMaximum-likelihood refinement was conducted in RELION using a weighted 3D CTF model.

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