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- EMDB-3015: Using recent advances in single-particle electron cryomicroscopy ... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-3015 | |||||||||
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Title | Using recent advances in single-particle electron cryomicroscopy structure determination for sub-tomogram averaging | |||||||||
![]() | sub-tomogram averaging reconstruction of the hepatitis B capsid. | |||||||||
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![]() | sub-tomogram averaging / RELION / maximum-likelihood / ultrastable gold substrates / hepatitis B capsid | |||||||||
Function / homology | ![]() 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 / viral envelope / structural molecule activity / DNA binding / RNA binding Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 8.1 Å | |||||||||
![]() | Bharat TA / Russo CJ / Lowe J / Passmore LA / Scheres SHW | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 9.1 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 8.9 KB 8.9 KB | Display Display | ![]() |
Images | ![]() | 80.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 221.1 KB | Display | ![]() |
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Full document | ![]() | 220.3 KB | Display | |
Data in XML | ![]() | 6.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | sub-tomogram averaging reconstruction of the hepatitis B capsid. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.17 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : hepatitis B capsid
Entire | Name: hepatitis B capsid |
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Components |
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-Supramolecule #1000: hepatitis B capsid
Supramolecule | Name: hepatitis B capsid / type: sample / ID: 1000 / Oligomeric state: icosahedral / Number unique components: 1 |
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Molecular weight | Theoretical: 3.8 MDa |
-Macromolecule #1: hepatitis B capsid
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 3.8 MDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Capsid protein |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | subtomogram averaging |
Aggregation state | particle |
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Sample preparation
Grid | Details: 300 mesh ultrastable gold substrates with 1.2 micron holes (Russo and Passmore, Science 2014) |
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: Gatan Quantum Energy Filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Details | Data was collected using SerialEM. |
Date | Aug 28, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 60 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.6 µm / Nominal defocus min: 3.3 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Details | Maximum-likelihood refinement was conducted in RELION using a weighted 3D CTF model. |
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Final reconstruction | Applied 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 |
CTF correction | Details: RELION 3D CTF Model |