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Yorodumi- EMDB-2095: Cryo-electron microscopy reconstruction of doublecortin-stabilise... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2095 | |||||||||
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Title | Cryo-electron microscopy reconstruction of doublecortin-stabilised microtubules in absence of kinesin | |||||||||
Map data | Reconstruction of doublecortin-stabilised microtubules in absence of kinesin | |||||||||
Sample |
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Keywords | doublecortin / microtubule / Microtubule-Associated Protein | |||||||||
Function / homology | Function and homology information axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / central nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / neuron migration / structural constituent of cytoskeleton / microtubule cytoskeleton organization ...axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / central nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / neuron migration / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / retina development in camera-type eye / mitotic cell cycle / nervous system development / microtubule binding / microtubule / cytoskeleton / intracellular signal transduction / hydrolase activity / neuron projection / protein heterodimerization activity / GTPase activity / GTP binding / protein kinase binding / metal ion binding / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | Bos taurus (cattle) / Homo sapiens (human) | |||||||||
Method | helical reconstruction / cryo EM / negative staining / Resolution: 8.3 Å | |||||||||
Authors | Liu JS / Schubert CR / Fu X / Fourniol FJ / Jaiswal JK / Houdusse A / Stultz CM / Moores CA / Walsh CA | |||||||||
Citation | Journal: Mol Cell / Year: 2012 Title: Molecular basis for specific regulation of neuronal kinesin-3 motors by doublecortin family proteins. Authors: Judy S Liu / Christian R Schubert / Xiaoqin Fu / Franck J Fourniol / Jyoti K Jaiswal / Anne Houdusse / Collin M Stultz / Carolyn A Moores / Christopher A Walsh / Abstract: Doublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of ...Doublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of Kif1a, a kinesin-3 motor protein that traffics synaptic vesicles. Neurons lacking Dcx and/or its structurally conserved paralogue, doublecortin-like kinase 1 (Dclk1), show impaired Kif1a-mediated transport of Vamp2, a cargo of Kif1a, with decreased run length. Human disease-associated mutations in Dcx's linker sequence (e.g., W146C, K174E) alter Kif1a/Vamp2 transport by disrupting Dcx/Kif1a interactions without affecting Dcx MT binding. Dcx specifically enhances binding of the ADP-bound Kif1a motor domain to MTs. Cryo-electron microscopy and subnanometer-resolution image reconstruction reveal the kinesin-dependent conformational variability of MT-bound Dcx and suggest a model for MAP-motor crosstalk on MTs. Alteration of kinesin run length by MAPs represents a previously undiscovered mode of control of kinesin transport and provides a mechanism for regulation of MT-based transport by local signals. | |||||||||
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_2095.map.gz | 1.3 MB | EMDB map data format | |
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Header (meta data) | emd-2095-v30.xml emd-2095.xml | 14.6 KB 14.6 KB | Display Display | EMDB header |
Images | 2095-image.png | 197.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2095 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2095 | HTTPS FTP |
-Validation report
Summary document | emd_2095_validation.pdf.gz | 255.1 KB | Display | EMDB validaton report |
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Full document | emd_2095_full_validation.pdf.gz | 254.2 KB | Display | |
Data in XML | emd_2095_validation.xml.gz | 4.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2095 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2095 | HTTPS FTP |
-Related structure data
Related structure data | 4atuMC 2098C 4atxC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2095.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of doublecortin-stabilised microtubules in absence of kinesin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 : Doublecortin-stabilised microtubules
Entire | Name: Doublecortin-stabilised microtubules |
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Components |
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-Supramolecule #1000: Doublecortin-stabilised microtubules
Supramolecule | Name: Doublecortin-stabilised microtubules / type: sample / ID: 1000 / Oligomeric state: 13-protofilament microtubule / Number unique components: 3 |
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-Macromolecule #1: tubulin alpha-1D chain
Macromolecule | Name: tubulin alpha-1D chain / type: protein_or_peptide / ID: 1 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Bos taurus (cattle) / synonym: cattle / Tissue: brain / Location in cell: cytoplasm |
Molecular weight | Theoretical: 50 KDa |
Sequence | InterPro: Alpha tubulin |
-Macromolecule #2: doublecortin
Macromolecule | Name: doublecortin / type: protein_or_peptide / ID: 2 / Name.synonym: DCX / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Theoretical: 40 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac |
Sequence | InterPro: Doublecortin domain |
-Macromolecule #3: tubulin beta-2B chain
Macromolecule | Name: tubulin beta-2B chain / type: protein_or_peptide / ID: 3 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Bos taurus (cattle) / synonym: cattle / Tissue: brain / Location in cell: cytoplasm |
Molecular weight | Theoretical: 50 KDa |
Sequence | InterPro: Beta tubulin, autoregulation binding site |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Buffer | pH: 6.8 Details: 20mM 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: FEI VITROBOT MARK I |
-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 |
Date | Oct 1, 2010 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Average electron dose: 17 e/Å2 / 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: 3.6 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | Particles were aligned using Spider and Frealign (Sindelar and Downing, 2010) |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 9.2 Å Applied symmetry - Helical parameters - Δ&Phi: 27.7 ° Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN Details: A sub-selection of approximately 146,000 tubulin dimers (75% of total dataset) went into the final reconstruction |
CTF correction | Details: done with FREALIGN |
-Atomic model buiding 1
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H |
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Software | Name: Chimera |
Details | Protocol: Rigid body |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation |
Output model | PDB-4atu: |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: Chimera, Flex-EM |
Details | Protocol: flexible fitting. The EM map was zoned around the doublecortin density. The N-DC atomic model (1MJD model 11, aa 38-150) was extended at its C-terminus (aa 151-156) using Chimera. Flexible fitting was performed into the zoned around EM map. The core DC domain (aa 53-129) and the docked W146 region (aa 145-146) were treated as a single rigid body. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross correlation |
Output model | PDB-4atu: |