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- PDB-4atx: Rigor kinesin motor domain with an ordered neck-linker, docked on... -

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Basic information

Entry
Database: PDB / ID: 4atx
TitleRigor kinesin motor domain with an ordered neck-linker, docked on tubulin dimer, modelled into the 8A cryo-EM map of doublecortin- microtubules decorated with kinesin
Components
  • KINESIN-1 HEAVY CHAIN
  • TUBULIN ALPHA-1D CHAIN
  • TUBULIN BETA-2B CHAIN
KeywordsHYDROLASE / MICROTUBULE / NECK-LINKER
Function / homology
Function and homology information


RHO GTPases activate KTN1 / Kinesins / regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / COPI-dependent Golgi-to-ER retrograde traffic / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / positive regulation of vesicle fusion / mitocytosis ...RHO GTPases activate KTN1 / Kinesins / regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / COPI-dependent Golgi-to-ER retrograde traffic / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / positive regulation of vesicle fusion / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / MHC class II antigen presentation / positive regulation of intracellular protein transport / ciliary rootlet / lysosome localization / positive regulation of potassium ion transport / JUN kinase binding / plus-end-directed microtubule motor activity / vesicle transport along microtubule / positive regulation of axon guidance / kinesin complex / microtubule motor activity / centrosome localization / mitochondrion transport along microtubule / microtubule lateral binding / stress granule disassembly / natural killer cell mediated cytotoxicity / synaptic vesicle transport / endocytic vesicle / positive regulation of insulin secretion involved in cellular response to glucose stimulus / postsynaptic cytosol / axonal growth cone / microtubule-based process / phagocytic vesicle / axon cytoplasm / dendrite cytoplasm / axon guidance / positive regulation of synaptic transmission, GABAergic / regulation of membrane potential / positive regulation of protein localization to plasma membrane / hippocampus development / cellular response to type II interferon / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / microtubule cytoskeleton / microtubule binding / vesicle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / neuron projection / hydrolase activity / protein heterodimerization activity / GTPase activity / GTP binding / protein-containing complex binding / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / metal ion binding / identical protein binding / cytoplasm
Similarity search - Function
Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. ...Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha-1D chain / Kinesin-1 heavy chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesRATTUS NORVEGICUS (Norway rat)
BOS TAURUS (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.2 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.
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.
#1: 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
DepositionMay 10, 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_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 1.2May 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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Assembly

Deposited unit
A: TUBULIN BETA-2B CHAIN
B: TUBULIN ALPHA-1D CHAIN
C: KINESIN-1 HEAVY CHAIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,2475
Polymers138,2813
Non-polymers9662
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.: 1 / 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.: 1 / 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 KINESIN-1 HEAVY CHAIN / CONVENTIONAL KINESIN HEAVY CHAIN / UBIQUITOUS KINESIN HEAVY CHAIN / UKHC


Mass: 38136.984 Da / Num. of mol.: 1 / Fragment: MOTOR DOMAIN, RESIDUES 1-340 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) RATTUS NORVEGICUS (Norway rat) / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: Q2PQA9, EC: 3.6.4.4
#4: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / 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.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM

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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 DECORATED WITH KINESIN MOTOR DOMAINS
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: Sep 1, 2009
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: 2900 nm / Nominal defocus min: 760 nm / Cs: 2 mm
Specimen holderTemperature: 93 K
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 63
Radiation wavelengthRelative weight: 1

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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: 8.2 Å / Num. of particles: 168000 / Nominal pixel size: 2.8 Å
Details: A HOMOLOGY MODEL OF RAT KINESIN MOTOR DOMAIN MUTANT T93N WAS GENERATED USING MODELLER, BASED ON THE STRUCTURE OF HUMAN KINESIN 1BG2. 2) KINESIN LOOP11 (AA 237-254) WAS OMITTED IN THIS MODEL ...Details: A HOMOLOGY MODEL OF RAT KINESIN MOTOR DOMAIN MUTANT T93N WAS GENERATED USING MODELLER, BASED ON THE STRUCTURE OF HUMAN KINESIN 1BG2. 2) KINESIN LOOP11 (AA 237-254) WAS OMITTED IN THIS MODEL AS NO CRYSTAL STRUCTURE REFLECTED THE CONFORMATION OF LOOP11 VISUALISED IN THE EM MAP SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2098. (DEPOSITION ID: 10790).
Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12XRP

2xrp

12XRP1PDBexperimental model
21BG2

1bg2

11BG22PDBexperimental model
RefinementHighest resolution: 8.2 Å
Refinement stepCycle: LAST / Highest resolution: 8.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9123 0 60 0 9183

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