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- EMDB-2095: Cryo-electron microscopy reconstruction of doublecortin-stabilise... -

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Basic information

Entry
Database: EMDB / ID: EMD-2095
TitleCryo-electron microscopy reconstruction of doublecortin-stabilised microtubules in absence of kinesin
Map dataReconstruction of doublecortin-stabilised microtubules in absence of kinesin
Sample
  • Sample: Doublecortin-stabilised microtubules
  • Protein or peptide: tubulin alpha-1D chain
  • Protein or peptide: doublecortin
  • Protein or peptide: tubulin beta-2B chain
Keywordsdoublecortin / 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 / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / nervous system development ...axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / central nervous system development / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / nervous system development / mitotic cell cycle / retina development in camera-type eye / microtubule cytoskeleton / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cytoskeleton / hydrolase activity / neuron projection / intracellular signal transduction / protein heterodimerization activity / GTPase activity / protein kinase binding / GTP binding / metal ion binding / cytoplasm / cytosol
Similarity search - Function
Neuronal migration protein doublecortin, chordata / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain / Doublecortin domain superfamily / Alpha tubulin / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. ...Neuronal migration protein doublecortin, chordata / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain / Doublecortin domain superfamily / Alpha tubulin / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Neuronal migration protein doublecortin / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (domestic cattle) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 8.3 Å
AuthorsLiu JS / Schubert CR / Fu X / Fourniol FJ / Jaiswal JK / Houdusse A / Stultz CM / Moores CA / Walsh CA
CitationJournal: 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
DepositionMay 9, 2012-
Header (metadata) releaseMay 17, 2012-
Map releaseAug 15, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4atu
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4atu
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2095-image.png

Downloads & links

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Map

FileDownload / File: emd_2095.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of doublecortin-stabilised microtubules in absence of kinesin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 101 pix.
= 282.8 Å		2.8 Å/pix.
x 41 pix.
= 114.8 Å		2.8 Å/pix.
x 85 pix.
= 238. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.6 / Movie #1: 1.6
Minimum - Maximum-8.86061192 - 12.475532530000001
Average (Standard dev.)0.57322699 (±2.0084734)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin506675
Dimensions4185101
Spacing4185101
CellA: 238.0 Å / B: 114.799995 Å / C: 282.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z8541101
origin x/y/z0.0000.0000.000
length x/y/z238.000114.800282.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS665075
NC/NR/NS8541101
D min/max/mean-8.86112.4760.573

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Supplemental data

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Sample components

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Entire : Doublecortin-stabilised microtubules

EntireName: Doublecortin-stabilised microtubules
Components
  • Sample: Doublecortin-stabilised microtubules
  • Protein or peptide: tubulin alpha-1D chain
  • Protein or peptide: doublecortin
  • Protein or peptide: tubulin beta-2B chain

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Supramolecule #1000: Doublecortin-stabilised microtubules

SupramoleculeName: 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

MacromoleculeName: tubulin alpha-1D chain / type: protein_or_peptide / ID: 1 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (domestic cattle) / synonym: cattle / Tissue: brain / Location in cell: cytoplasm
Molecular weightTheoretical: 50 KDa
SequenceInterPro: Alpha tubulin

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Macromolecule #2: doublecortin

MacromoleculeName: doublecortin / type: protein_or_peptide / ID: 2 / Name.synonym: DCX / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac
SequenceInterPro: Doublecortin domain

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Macromolecule #3: tubulin beta-2B chain

MacromoleculeName: tubulin beta-2B chain / type: protein_or_peptide / ID: 3 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (domestic cattle) / synonym: cattle / Tissue: brain / Location in cell: cytoplasm
Molecular weightTheoretical: 50 KDa
SequenceInterPro: Beta tubulin, autoregulation binding site

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Details: 20mM PIPES, 1mM EGTA, 3mM MgCl2, 1mM TCEP, 0.5mM GTP
StainingType: NEGATIVE / Details: cryo-EM
GridDetails: 300 mesh lacey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateOct 1, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Average electron dose: 17 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles were aligned using Spider and Frealign (Sindelar and Downing, 2010)
Final reconstructionApplied 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 correctionDetails: done with FREALIGN

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Atomic model buiding 1

Initial modelPDB ID:

2xrp


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
SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

PDB-4atu:
Human doublecortin N-DC repeat plus linker, and tubulin (2XRP) docked into an 8A cryo-EM map of doublecortin-stabilised microtubules reconstructed in absence of kinesin

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Atomic model buiding 2

Initial modelPDB ID:

1mjd

SoftwareName: Chimera, Flex-EM
DetailsProtocol: 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross correlation
Output model

PDB-4atu:
Human doublecortin N-DC repeat plus linker, and tubulin (2XRP) docked into an 8A cryo-EM map of doublecortin-stabilised microtubules reconstructed in absence of kinesin

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