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

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Entry
Database: EMDB / ID: 2095
TitleCryo-electron microscopy reconstruction of doublecortin-stabilised microtubules in absence of kinesin
Map dataReconstruction of doublecortin-stabilised microtubules in absence of kinesin
SampleDoublecortin-stabilised microtubules:
tubulin alpha-1D chain / doublecortin / tubulin beta-2B chain
Keywordsdoublecortin / microtubule / Microtubule-Associated Protein
Function / homologyTubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, C-terminal domain superfamily / Doublecortin domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Neuronal migration protein doublecortin, chordata / Tubulin, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, C-terminal / Recycling pathway of L1 ...Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, C-terminal domain superfamily / Doublecortin domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Neuronal migration protein doublecortin, chordata / Tubulin, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, C-terminal / Recycling pathway of L1 / Doublecortin domain superfamily / Tubulin/FtsZ family, GTPase domain / Beta tubulin / Doublecortin / Tubulin C-terminal domain / Tubulin subunits alpha, beta, and gamma signature. / Tubulin-beta mRNA autoregulation signal. / Doublecortin domain profile. / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / MHC class II antigen presentation / Separation of Sister Chromatids / Resolution of Sister Chromatid Cohesion / Tubulin/FtsZ, GTPase domain / Alpha tubulin / Recruitment of NuMA to mitotic centrosomes / Tubulin / Neurofascin interactions / Kinesins / Carboxyterminal post-translational modifications of tubulin / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases Activate Formins / Hedgehog 'on' state / RHO GTPases activate IQGAPs / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Doublecortin domain / positive regulation of axon guidance / microtubule associated complex / Beta tubulin, autoregulation binding site / Alpha tubulin / microtubule-based process / neuron migration / structural constituent of cytoskeleton / central nervous system development / microtubule cytoskeleton / nervous system development / microtubule / microtubule binding / neuron projection / cytoskeleton / intracellular signal transduction / GTPase activity / GTP binding / protein kinase binding / protein heterodimerization activity / cytosol / cytoplasm / Neuronal migration protein doublecortin / Tubulin alpha-1D chain / Tubulin beta-2B chain
Function and homology information
SourceBos taurus (cattle) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / 8.3 Å resolution
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.
Validation ReportPDB-ID: 4atu

SummaryFull reportAbout validation report
DateDeposition: May 9, 2012 / Header (metadata) release: May 17, 2012 / Map release: Aug 15, 2012 / Last update: Sep 26, 2012

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Structure visualization

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

Downloads & links

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Map

Fileemd_2095.map.gz (map file in CCP4 format, 1376 KB)
Projections & slices

Image control

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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density
Contour Level:1.6 (by author), 1.6 (movie #1):
Minimum - Maximum-8.86061192 - 12.47553253
Average (Standard dev.)0.57322699 (2.0084734)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions4185101
Origin506675
Limit90150175
Spacing4185101
CellA: 238 Å / B: 114.799995 Å / C: 282.8 Å
α=β=γ: 90 deg.

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 / Number of components: 3 / Oligomeric State: 13-protofilament microtubule

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Component #1: protein, tubulin alpha-1D chain

ProteinName: tubulin alpha-1D chain / Oligomeric Details: heterodimer / Recombinant expression: No
MassTheoretical: 50 kDa
SourceSpecies: Bos taurus (cattle)
Source (natural)Location in cell: cytoplasm / Organ or tissue: brain
External referencesInterPro: Alpha tubulin

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Component #2: protein, doublecortin

ProteinName: doublecortin / a.k.a: DCX / Recombinant expression: Yes
MassTheoretical: 40 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Vector: pFastBac
External referencesInterPro: Doublecortin domain

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Component #3: protein, tubulin beta-2B chain

ProteinName: tubulin beta-2B chain / Oligomeric Details: heterodimer / Recombinant expression: No
MassTheoretical: 50 kDa
SourceSpecies: Bos taurus (cattle)
Source (natural)Location in cell: cytoplasm / Organ or tissue: brain
External referencesInterPro: Beta tubulin, autoregulation binding site

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Experimental details

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Sample preparation

SpecimenSpecimen state: filament / Method: cryo EM
Helical parametersHand: LEFT HANDED / Delta z: 9.2 Å / Delta phi: 27.7 deg.
Sample solutionBuffer solution: 20mM PIPES, 1mM EGTA, 3mM MgCl2, 1mM TCEP, 0.5mM GTP
pH: 6.8
Support film300 mesh lacey carbon grids
Stainingcryo-EM
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Oct 1, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 17 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal)
Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 900 - 3600 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC / Temperature: 93 K
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionScanner: ZEISS SCAI / Bit depth: 8

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Image processing

ProcessingMethod: helical reconstruction
Details: Particles were aligned using Spider and Frealign (Sindelar and Downing, 2010)
3D reconstructionAlgorithm: Single particle / Software: SPIDER, FREALIGN / CTF correction: done with FREALIGN
Details: A sub-selection of approximately 146,000 tubulin dimers (75% of total dataset) went into the final reconstruction
Resolution: 8.3 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Target criteria: cross correlation / Refinement space: REAL / Details: Protocol: Rigid body
Input PDB model: 2XRP
Chain ID: A, B, C, D, E, F, G, H
Modeling #2Software: Chimera, Flex-EM / Refinement protocol: flexible / Target criteria: cross correlation / Refinement space: REAL
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.
Input PDB model: 1MJD
Output model

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