+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6513 | |||||||||
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Title | 3D reconstruction of tarantula thick filaments | |||||||||
Map data | 3D reconstruction of tarantula thick filament at 17 Angstrom resolution | |||||||||
Sample |
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Keywords | single particle analysis / thick filament / muscle contraction | |||||||||
Biological species | Grammostola rosea (Chilean rose tarantula) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 17.0 Å | |||||||||
Authors | Yang S / Woodhead JL / Zhao F / Sulbaran G / Craig R | |||||||||
Citation | Journal: J Struct Biol / Year: 2016 Title: An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits. Authors: Shixin Yang / John L Woodhead / Fa-Qing Zhao / Guidenn Sulbarán / Roger Craig / Abstract: Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a ...Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a challenge to achieve high resolution for filaments with flexible subunits and a large axial rise per subunit relative to pixel size. Here, we describe an approach that improves the resolution in such cases. In filaments with a large axial rise, many segments must be shifted a long distance along the filament axis to match with a reference projection, potentially causing loss of alignment accuracy and hence resolution. In our study of myosin filaments, we overcame this problem by pre-determining the axial positions of myosin head crowns within segments to decrease the alignment error. In addition, homogeneous, well-ordered segments were selected from the raw data set by checking the assigned azimuthal rotation angle of segments in each filament against those expected for perfect helical symmetry. These procedures improved the resolution of the filament reconstruction from 30 Å to 13 Å. This approach could be useful in other helical filaments with a large axial rise and/or flexible subunits. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6513.map.gz | 49.4 MB | EMDB map data format | |
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Header (meta data) | emd-6513-v30.xml emd-6513.xml | 9.8 KB 9.8 KB | Display Display | EMDB header |
Images | 400_6513.gif 80_6513.gif | 73 KB 5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6513 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6513 | HTTPS FTP |
-Validation report
Summary document | emd_6513_validation.pdf.gz | 79.1 KB | Display | EMDB validaton report |
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Full document | emd_6513_full_validation.pdf.gz | 78.2 KB | Display | |
Data in XML | emd_6513_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6513 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6513 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6513.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D reconstruction of tarantula thick filament at 17 Angstrom resolution | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.744 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : tarantula thick filament
Entire | Name: tarantula thick filament |
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Components |
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-Supramolecule #1000: tarantula thick filament
Supramolecule | Name: tarantula thick filament / type: sample / ID: 1000 / Oligomeric state: helical filaments of myosin II / Number unique components: 1 |
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Molecular weight | Experimental: 520 KDa / Theoretical: 520 KDa / Method: Sedimentation |
-Macromolecule #1: thick filament
Macromolecule | Name: thick filament / type: protein_or_peptide / ID: 1 / Name.synonym: myosin filament / Number of copies: 18 / Oligomeric state: helical filament / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Grammostola rosea (Chilean rose tarantula) / synonym: tarantula / Tissue: leg / Cell: striated muscle / Organelle: myofibril / Location in cell: cytoskeleton |
Molecular weight | Experimental: 520 KDa / Theoretical: 520 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 1.0 mg/mL |
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Buffer | pH: 7 Details: 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 mM PIPES, 5 mM NaH2PO4, 1 mM NaN3 |
Grid | Details: Quantifoil (R2/2) 200 mesh grids, glow discharged |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 85 K / Instrument: HOMEMADE PLUNGER / Method: Blot for 2 seconds before plunging |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Date | Apr 13, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 690 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 109000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder: Nitrogen-cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Cryo-EM images of tarantula thick filaments were processed using SPIDER and IHRSR |
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CTF correction | Details: each particle |
Final reconstruction | Applied symmetry - Helical parameters - Δz: 145 Å Applied symmetry - Helical parameters - Δ&Phi: 30 ° Applied symmetry - Helical parameters - Axial symmetry: C4 (4 fold cyclic) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: SPIDER, IHRSR Details: Particles were pre-centered and all particles are included in reconstruction. Number images used: 3174 |