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- PDB-4ck7: Pseudo-atomic model of microtubule-bound human kinesin-5 motor do... -

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Basic information

Entry
Database: PDB / ID: 4ck7
TitlePseudo-atomic model of microtubule-bound human kinesin-5 motor domain in presence of adp.alfx (NECK-LINKER IN ITS DISCONNECTED CONFORMATION, 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


Kinesins / plus-end-directed microtubule motor activity / positive regulation of axon guidance / regulation of mitotic centrosome separation / COPI-dependent Golgi-to-ER retrograde traffic / mitotic centrosome separation / microtubule motor activity / kinesin complex / spindle organization / mitotic spindle assembly ...Kinesins / plus-end-directed microtubule motor activity / positive regulation of axon guidance / regulation of mitotic centrosome separation / COPI-dependent Golgi-to-ER retrograde traffic / mitotic centrosome separation / microtubule motor activity / kinesin complex / spindle organization / mitotic spindle assembly / microtubule-based movement / microtubule-based process / MHC class II antigen presentation / mitotic spindle organization / mitotic spindle / microtubule cytoskeleton organization / structural constituent of cytoskeleton / microtubule cytoskeleton / spindle / spindle pole / nervous system development / mitotic cell cycle / microtubule binding / microtubule / protein heterodimerization activity / cell division / GTP binding / protein kinase binding / protein-containing complex / membrane / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Kinesin-associated microtubule-binding / Kinesin-associated microtubule-binding domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor domain / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site ...Kinesin-associated microtubule-binding / Kinesin-associated microtubule-binding domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor domain / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Alpha tubulin / Kinesin motor domain superfamily / Tubulin C-terminal domain / Tubulin, C-terminal / Tubulin / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal / 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
Kinesin-like protein KIF11 / Tubulin alpha-1D chain / ADENOSINE-5'-DIPHOSPHATE / ALUMINUM FLUORIDE / TAXOL / GUANOSINE-5'-TRIPHOSPHATE / GUANOSINE-5'-DIPHOSPHATE / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
BOS TAURUS (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.2 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

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-2533
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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)144,19710
Polymers141,8173
Non-polymers2,3807
Water0
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 (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 (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 PROTEIN 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 , 6 types, 7 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#6: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#7: Chemical ChemComp-TA1 / TAXOL / Paclitaxel


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM
#8: Chemical ChemComp-AF3 / ALUMINUM FLUORIDE / Aluminium fluoride


Mass: 83.977 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF3
#9: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine 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 / Type: COMPLEX
Buffer solutionName: 80 MM PIPES 5 MM MGCL2 1 MM EGTA 1 MM TCEP / pH: 6.8 / Details: 80 MM PIPES 5 MM MGCL2 1 MM EGTA 1 MM TCEP
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: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Mar 10, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm / Cs: 2 mm
Specimen holderTemperature: 90 K
Image recordingElectron dose: 18 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 125

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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: 9.2 Å / Num. of particles: 10692
Details: THE N-TERMINAL SEQUENCE WAS MODELLED USING MODELLER. THE DISCONNECTED CONFORMATION OF THE NECK-LINKER WAS CALCULATED USING A CONJUGATE-GRADIENT ENERGY MINIMIZATION. SUBMISSION BASED ON ...Details: THE N-TERMINAL SEQUENCE WAS MODELLED USING MODELLER. THE DISCONNECTED CONFORMATION OF THE NECK-LINKER WAS CALCULATED USING A CONJUGATE-GRADIENT ENERGY MINIMIZATION. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2533. (DEPOSITION ID: 12198).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: METHOD--FLEXIBLE REFINEMENT PROTOCOL--X-RAY
Atomic model building
IDPDB-ID 3D fitting-ID
13HQD1
21JFF1
RefinementHighest resolution: 9.2 Å
Refinement stepCycle: LAST / Highest resolution: 9.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1199 0 155 0 1354

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