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- PDB-4ck5: Pseudo-atomic model of microtubule-bound human kinesin-5 motor do... -

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Basic information

Entry
Database: PDB / ID: 4ck5
TitlePseudo-atomic model of microtubule-bound human kinesin-5 motor domain in the ADP state, based on cryo-electron microscopy experiment.
Components
  • KINESIN-LIKE PROTEIN KIF11
  • TUBULIN ALPHA-1D CHAIN
  • TUBULIN BETA-2B CHAIN
KeywordsMOTOR PROTEIN / KINESINS / MICROTUBULES / MITOSIS / MECHANOCHEMISTRY
Function / homology
Function and homology information


spindle elongation / regulation of mitotic centrosome separation / plus-end-directed microtubule motor activity / Kinesins / mitotic centrosome separation / positive regulation of axon guidance / kinesin complex / microtubule motor activity / spindle organization / COPI-dependent Golgi-to-ER retrograde traffic ...spindle elongation / regulation of mitotic centrosome separation / plus-end-directed microtubule motor activity / Kinesins / mitotic centrosome separation / positive regulation of axon guidance / kinesin complex / microtubule motor activity / spindle organization / COPI-dependent Golgi-to-ER retrograde traffic / microtubule-based movement / mitotic spindle assembly / microtubule-based process / MHC class II antigen presentation / mitotic spindle organization / structural constituent of cytoskeleton / microtubule cytoskeleton organization / spindle / neuron migration / spindle pole / mitotic spindle / mitotic cell cycle / microtubule cytoskeleton / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / ciliary basal body / protein heterodimerization activity / cell division / intracellular membrane-bounded organelle / GTPase activity / protein kinase binding / GTP binding / protein-containing complex / ATP binding / metal ion binding / nucleus / membrane / cytoplasm / cytosol
Similarity search - Function
Kinesin-associated microtubule-binding domain / Kinesin-associated microtubule-binding / : / : / 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-associated microtubule-binding domain / Kinesin-associated microtubule-binding / : / : / 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
ADENOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Kinesin-like protein KIF11 / 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: 10 Å
Model type detailsCA ATOMS ONLY, CHAIN A, B, C
AuthorsGoulet, A. / Major, J. / Jun, Y. / Gross, S. / Rosenfeld, S. / Moores, C.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family.
Authors: Adeline Goulet / Jennifer Major / Yonggun Jun / Steven P Gross / Steven S Rosenfeld / Carolyn A Moores /
Abstract: Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and ...Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin--the transport motor kinesin-1--is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor's structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.
History
DepositionDec 30, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2014Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2014Group: Database references
Revision 1.2Aug 30, 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 1.3May 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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  • Simplified surface model + fitted atomic model
  • EMDB-2537
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Structure viewerMolecule:
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Assembly

Deposited unit
A: TUBULIN ALPHA-1D CHAIN
B: TUBULIN BETA-2B CHAIN
C: KINESIN-LIKE PROTEIN KIF11
hetero molecules


Theoretical massNumber of molelcules
Total (without water)144,0898
Polymers141,8173
Non-polymers2,2725
Water00
1


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

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein TUBULIN ALPHA-1D CHAIN / ALPHA TUBULIN / Coordinate model: Cα atoms only


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
#2: Protein TUBULIN BETA-2B CHAIN / BETA TUBULIN / Coordinate model: Cα atoms only


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
#3: Protein KINESIN-LIKE PROTEIN KIF11 / KINESIN-LIKE PROTEIN 1 / KINESIN-LIKE SPINDLE PROTEIN HKSP / KINESIN-RELATED MOTOR PROTEIN EG5 / ...KINESIN-LIKE PROTEIN 1 / KINESIN-LIKE SPINDLE PROTEIN HKSP / KINESIN-RELATED MOTOR PROTEIN EG5 / THYROID RECEPTOR-INTERACTING PR OTEIN 5 / TR-INTERACTING PROTEIN 5 / TRIP-5 / KINESIN-5 / Coordinate model: Cα atoms only


Mass: 41673.105 Da / Num. of mol.: 1 / Fragment: MOTOR DOMAIN, RESIDUES 1-367 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: PET21A / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 / References: UniProt: P52732

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Non-polymers , 5 types, 5 molecules

#4: 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
#5: 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
#6: Chemical ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Details

Sequence detailsCYS-LITE MUTANT CONTAINING THE SUBSTITUTION T126C

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: microtubule-bound human kinesin-5 motor domain in the ADP state
Type: COMPLEX
Details: 13-PROTOFILAMENT MICROTUBULE- BOUND HUMAN KINESIN-5 MOTOR DOMAIN IN PRESENCE OF ADP
Buffer solutionName: 20 MM PIPES, 5 MM MGCL2, 1 MM EGTA, 10 MM ADP / pH: 6.8 / Details: 20 MM PIPES, 5 MM MGCL2, 1 MM EGTA, 10 MM ADP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Oct 20, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 68000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 700 nm / Cs: 2 mm
Specimen holderTemperature: 90 K
Image recordingElectron dose: 18 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 160

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Processing

EM software
IDNameCategory
1Flex-EMmodel fitting
2UCSF Chimeramodel fitting
3FREALIGN3D reconstruction
4SPIDER3D reconstruction
CTF correctionDetails: FREALIGN
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 10 Å / Num. of particles: 9615 / Actual pixel size: 2.2 Å
Details: TO ACCOUNT FOR THE FACT THAT HELIX ALPHA4 IS LONGER IN OUR RECONSTRUCTION THAN IN THE AVAILABLE ADP CRYSTAL STRUCTURES, WE MODELLED 7 HELICAL TURNS OF HELIX ALPHA4 ( RESIDUES D279-E304), AS ...Details: TO ACCOUNT FOR THE FACT THAT HELIX ALPHA4 IS LONGER IN OUR RECONSTRUCTION THAN IN THE AVAILABLE ADP CRYSTAL STRUCTURES, WE MODELLED 7 HELICAL TURNS OF HELIX ALPHA4 ( RESIDUES D279-E304), AS OBSERVED IN THE ENTRY 3HQD. THE N- TERMINAL SEQUENCE WAS MODELLED USING MODELLER. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2537. (DEPOSITION ID: 12202).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: METHOD--FLEXIBLE
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13HQD

3hqd

13HQD1PDBexperimental model
21JFF

1jff

11JFF2PDBexperimental model
RefinementHighest resolution: 10 Å
Refinement stepCycle: LAST / Highest resolution: 10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1188 0 150 0 1338

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