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- PDB-4atu: Human doublecortin N-DC repeat plus linker, and tubulin (2XRP) do... -

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Basic information

Entry
Database: PDB / ID: 4atu
TitleHuman 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
Components
  • NEURONAL MIGRATION PROTEIN DOUBLECORTIN
  • TUBULIN ALPHA-1D CHAIN
  • TUBULIN BETA-2B CHAIN
KeywordsHYDROLASE / 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 / neuron projection / intracellular signal transduction / hydrolase activity / 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 superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin ...Neuronal migration protein doublecortin, chordata / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Neuronal migration protein doublecortin / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
BOS TAURUS (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.3 Å
AuthorsLiu, J.S. / Schubert, C.R. / Fu, X. / Fourniol, F.J. / Jaiswal, J.K. / Houdusse, A. / Stultz, C.M. / Moores, C.A. / Walsh, C.A.
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, 2012Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 26, 2012Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_image_scans / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 1.2Jun 20, 2018Group: Data collection / Derived calculations / Category: struct_conn
Revision 1.3May 8, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: TUBULIN BETA-2B CHAIN
B: TUBULIN ALPHA-1D CHAIN
C: TUBULIN BETA-2B CHAIN
D: TUBULIN ALPHA-1D CHAIN
E: TUBULIN BETA-2B CHAIN
F: TUBULIN ALPHA-1D CHAIN
G: TUBULIN BETA-2B CHAIN
H: TUBULIN ALPHA-1D CHAIN
I: NEURONAL MIGRATION PROTEIN DOUBLECORTIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)446,04717
Polymers442,1819
Non-polymers3,8668
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein
TUBULIN BETA-2B CHAIN


Mass: 49907.770 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) BOS TAURUS (domestic cattle) / Organ: BRAIN / References: UniProt: Q6B856, EC: 3.6.5.6
#2: Protein
TUBULIN ALPHA-1D CHAIN


Mass: 50236.352 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) BOS TAURUS (domestic cattle) / Organ: BRAIN / References: UniProt: Q2HJ86, EC: 3.6.5.6
#3: Protein NEURONAL MIGRATION PROTEIN DOUBLECORTIN / DOUBLIN / LISSENCEPHALIN-X / LIS-X


Mass: 41604.727 Da / Num. of mol.: 1 / Fragment: RESIDUES 2-360
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: PFASTBAC / Cell line (production host): SF9 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: O43602
#4: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#5: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
Sequence detailsFULL-LENGTH HUMAN DOUBLECORTIN ISOFORM 2ENGINEERED WITH AN N- TERMINAL FLAG TAG

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: DOUBLECORTIN-STABILISED MICROTUBULES / Type: COMPLEX
Buffer solutionName: 20MM PIPES, 1MM EGTA, 3MM MGCL2, 1MM TCEP, 0.5MM GTP / pH: 6.8
Details: 20MM PIPES, 1MM EGTA, 3MM MGCL2, 1MM TCEP, 0.5MM GTP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE
Details: CRYOGEN- ETHANE, HUMIDITY- 100, INSTRUMENT- FEI VITROBOT

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Oct 1, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 50000 X / Nominal defocus max: 3600 nm / Nominal defocus min: 900 nm / Cs: 2 mm
Specimen holderTemperature: 93 K
Image recordingElectron dose: 17 e/Å2 / Film or detector model: KODAK SO-163 FILM

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Processing

EM software
IDNameCategory
1Flex-EMmodel fitting
2UCSF Chimeramodel fitting
3FREALIGN3D reconstruction
4SPIDER3D reconstruction
CTF correctionDetails: DONE WITH FREALIGN
3D reconstructionMethod: SINGLE PARTICLE / Resolution: 8.3 Å / Num. of particles: 146000 / Nominal pixel size: 2.8 Å
Details: DOUBLECORTIN LINKER REGION 151-156 WAS MODELLED INTO THE EM MAP USING CHIMERA AND REFINED USING FLEX-EM SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2095. (DEPOSITION ID: 10785).
Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY
Atomic model buildingPDB-ID: 2XRP

2xrp


Accession code: 2XRP / Source name: PDB / Type: experimental model
RefinementHighest resolution: 8.3 Å
Refinement stepCycle: LAST / Highest resolution: 8.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms27600 0 240 0 27840

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