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Yorodumi- EMDB-1787: Asymmetric reconstruction of doublecortin-stabilised microtubules... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1787 | |||||||||
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Title | Asymmetric reconstruction of doublecortin-stabilised microtubules decorated with kinesin motor domain | |||||||||
Map data | This is a C1 reconstruction of doublecortin-stabilised microtubule decorated with kinesin motor domain | |||||||||
Sample |
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Keywords | Tubulin / MAP / microtubule / stabilisation / doublecortin / kinesin | |||||||||
Biological species | Bos taurus (cattle) / Homo sapiens (human) / Rattus norvegicus (Norway rat) | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 13.5 Å | |||||||||
Authors | Fourniol FJ / Sindelar CV / Amigues B / Clare D / Thomas G / Perderiset M / Francis F / Houdusse A / Moores CA | |||||||||
Citation | Journal: J Cell Biol / Year: 2010 Title: Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution. Authors: Franck J Fourniol / Charles V Sindelar / Béatrice Amigues / Daniel K Clare / Geraint Thomas / Mylène Perderiset / Fiona Francis / Anne Houdusse / Carolyn A Moores / Abstract: Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed ...Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX's unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin-tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX's exquisite binding selectivity uncovers important insights into regulation of cellular MTs. | |||||||||
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_1787.map.gz | 28 MB | EMDB map data format | |
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Header (meta data) | emd-1787-v30.xml emd-1787.xml | 11.6 KB 11.6 KB | Display Display | EMDB header |
Images | 1787_image.png | 429.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1787 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1787 | HTTPS FTP |
-Validation report
Summary document | emd_1787_validation.pdf.gz | 263.9 KB | Display | EMDB validaton report |
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Full document | emd_1787_full_validation.pdf.gz | 263 KB | Display | |
Data in XML | emd_1787_validation.xml.gz | 6.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1787 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1787 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1787.map.gz / Format: CCP4 / Size: 37.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | This is a C1 reconstruction of doublecortin-stabilised microtubule decorated with kinesin motor domain | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 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 : Microtubules co-polymerised with doublecortin and bound with kine...
Entire | Name: Microtubules co-polymerised with doublecortin and bound with kinesin motor domain |
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Components |
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-Supramolecule #1000: Microtubules co-polymerised with doublecortin and bound with kine...
Supramolecule | Name: Microtubules co-polymerised with doublecortin and bound with kinesin motor domain type: sample / ID: 1000 / Oligomeric state: 13-protofilament microtubule / Number unique components: 3 |
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-Macromolecule #1: Alpha-beta tubulin dimer
Macromolecule | Name: Alpha-beta tubulin dimer / type: protein_or_peptide / ID: 1 / Name.synonym: Tubulin dimer / 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: Cytoplasm |
-Macromolecule #2: Doublecortin
Macromolecule | Name: Doublecortin / type: protein_or_peptide / ID: 2 / Name.synonym: DCX / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac |
-Macromolecule #3: Kinesin motor domain
Macromolecule | Name: Kinesin motor domain / type: protein_or_peptide / ID: 3 / Name.synonym: Kinesin head / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) / synonym: Rat / Location in cell: Cytoplasm |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 6.8 Details: 80mM Pipes, 1mM EGTA, 3mM MgCl2, 1mM TCEP, 0.5mM GTP |
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Staining | Type: NEGATIVE / Details: cryo-EM |
Grid | Details: 300 mesh lacey carbon grids |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot (FEI) / Method: Chamber at 37 degrees C, blot 2s |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Average: 93 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 63 / Average electron dose: 15 e/Å2 / Details: Images were binned with a factor of 2 / Bits/pixel: 8 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.76 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: Eucentric / Specimen holder model: OTHER |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: FREALIGN |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN Details: C1 map calculated from approximately 13,000 one-dimer-long microtubule segments |