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Yorodumi- EMDB-5489: Improved Reconstruction of the Human Ndc80 Bonsai Decorated Micro... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5489 | |||||||||
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Title | Improved Reconstruction of the Human Ndc80 Bonsai Decorated Microtubule | |||||||||
Map data | Reconstruction of 14 protofilament microtubule decorated with Ndc80 Bonsai | |||||||||
Sample |
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Keywords | microtubule / Ndc80 / Hec1 / kinetochore / mitosis | |||||||||
Biological species | Bos taurus (cattle) / Homo sapiens (human) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 7.9 Å | |||||||||
Authors | Alushin GM / Musinipally V / Matson D / Tooley J / Stukenberg PT / Nogales E | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2012 Title: Multimodal microtubule binding by the Ndc80 kinetochore complex. Authors: Gregory M Alushin / Vivek Musinipally / Daniel Matson / John Tooley / P Todd Stukenberg / Eva Nogales / Abstract: The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular 'head' formed by tandem calponin-homology domains ...The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular 'head' formed by tandem calponin-homology domains and an 80-amino-acid unstructured 'tail' that contains sites of phosphoregulation by the Aurora B kinase. Using biochemical, cell biological and electron microscopy analyses, we dissected the roles of the tail in binding of microtubules and mediation of cooperative interactions between Ndc80 complexes. Two segments of the tail that contain Aurora B phosphorylation sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions that are disrupted by phosphorylation. We propose a model in which Ndc80's interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail. | |||||||||
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_5489.map.gz | 55.1 MB | EMDB map data format | |
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Header (meta data) | emd-5489-v30.xml emd-5489.xml | 13 KB 13 KB | Display Display | EMDB header |
Images | emd_5489.png | 236.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5489 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5489 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5489.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of 14 protofilament microtubule decorated with Ndc80 Bonsai | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.48 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 : Human Ndc80 bonsai complex bound to the microtubule
Entire | Name: Human Ndc80 bonsai complex bound to the microtubule |
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Components |
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-Supramolecule #1000: Human Ndc80 bonsai complex bound to the microtubule
Supramolecule | Name: Human Ndc80 bonsai complex bound to the microtubule / type: sample / ID: 1000 Details: Ndc80 bonsai is a heterodimer of Ndc80-Spc25 and Nuf2-Spc24; tubulin is a heterodimer of alpha and beta tubulin Oligomeric state: 2 copies of the Ndc80 bonsai complex bind to each tubulin heterodimer Number unique components: 2 |
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Molecular weight | Theoretical: 260 KDa |
-Macromolecule #1: tubulin
Macromolecule | Name: tubulin / type: protein_or_peptide / ID: 1 / Name.synonym: tubulin / Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Bos taurus (cattle) / synonym: bovine / Tissue: brain / Location in cell: cytoskeleton |
Molecular weight | Theoretical: 110 KDa |
-Macromolecule #2: Ndc80-Spc25 Chimera
Macromolecule | Name: Ndc80-Spc25 Chimera / type: protein_or_peptide / ID: 2 Details: Chimera of Ndc80 residues 1-286 and Spc25 residues 118-224 Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: human / Organelle: Nucleus / Location in cell: Kinetochore |
Molecular weight | Theoretical: 45 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX6p-2RBS |
-Macromolecule #3: Nuf2-Spc24 Chimera
Macromolecule | Name: Nuf2-Spc24 Chimera / type: protein_or_peptide / ID: 3 Details: Chimera of Nuf2 residues 1-169 and Spc24 residues 122-197 Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Organelle: Nucleus / Location in cell: Kinetochore |
Molecular weight | Theoretical: 29 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX6p-2RBS |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 0.25 mg/mL |
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Buffer | pH: 6.8 Details: 80mM PIPES, 1mM MgCl2, 1mM EGTA, 1mM DTT, 0.05% Nonidet P-40, 20uM taxol |
Grid | Details: C-flat 1.2/1.3 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK II Method: 2 uL of 0.25 mg/mL MTs applied to grid for 1 minute, 4 uL of 0.7 mg/mL Ndc80 bonsai added, manually blot 1 minute, another 4 uL of Ndc80 applied for 1 minute, 2 uL removed with pipettor, blot ...Method: 2 uL of 0.25 mg/mL MTs applied to grid for 1 minute, 4 uL of 0.7 mg/mL Ndc80 bonsai added, manually blot 1 minute, another 4 uL of Ndc80 applied for 1 minute, 2 uL removed with pipettor, blot for 2 seconds before plunging, 0 mm offset |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Alignment procedure | Legacy - Astigmatism: objective lens astigmatism corrected at 100Kx mag |
Date | Mar 12, 2009 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 100 / Average electron dose: 15 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: side-entry / Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | The particles were aligned using IHRSR in EMAN2/SPARX followed by FREALIGN |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 8.59601 Å Applied symmetry - Helical parameters - Δ&Phi: 25.74778 ° Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2/SPARX, FREALIGN Details: Particles were initially aligned using multi-model IHRSR protocol in EMAN2/SPARX with naked 13 and 14 protofilament microtubules as references. Final reconstruction and CTF correction were ...Details: Particles were initially aligned using multi-model IHRSR protocol in EMAN2/SPARX with naked 13 and 14 protofilament microtubules as references. Final reconstruction and CTF correction were performed for 14 protofilament segments with FREALIGN v8.9. A B-factor of -1000 was applied with BFACTOR with a peak at 8.3 Angstrom. FSC was calculated only for MT and Ndc80-NUF2 head, disordered outer head was excluded with soft mask. |
CTF correction | Details: Each particle |