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- PDB-4aqw: Model of human kinesin-5 motor domain (1II6, 3HQD) and mammalian ... -

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Basic information

Entry
Database: PDB / ID: 4aqw
TitleModel of human kinesin-5 motor domain (1II6, 3HQD) and mammalian tubulin heterodimer (1JFF) docked into the 9.5-angstrom cryo-EM map of microtubule-bound kinesin-5 motor domain in the rigor state.
Components
  • KINESIN-LIKE PROTEIN KIF11
  • TUBULIN ALPHA-1D CHAIN
  • TUBULIN BETA-2B CHAIN
KeywordsMOTOR PROTEIN / MICROTUBULE / MITOSIS / CANCER
Function / homology
Function and homology information


spindle elongation / regulation of mitotic centrosome separation / Kinesins / mitotic centrosome separation / plus-end-directed microtubule motor activity / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / microtubule motor activity / spindle organization / kinesin complex ...spindle elongation / regulation of mitotic centrosome separation / Kinesins / mitotic centrosome separation / plus-end-directed microtubule motor activity / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / microtubule motor activity / spindle organization / kinesin complex / microtubule-based movement / microtubule-based process / mitotic spindle assembly / MHC class II antigen presentation / mitotic spindle organization / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / spindle pole / spindle / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / protein heterodimerization activity / cell division / GTPase activity / GTP binding / protein kinase binding / protein-containing complex / ATP binding / membrane / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Kinesin-associated microtubule-binding domain / Kinesin-associated microtubule-binding / : / : / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain ...Kinesin-associated microtubule-binding domain / Kinesin-associated microtubule-binding / : / : / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / 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 / TAXOL / Kinesin-like protein KIF11 / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
BOS TAURUS (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.5 Å
Model type detailsCA ATOMS ONLY, CHAIN A, B, C
AuthorsGoulet, A. / Behnke-Parks, W.M. / Sindelar, C.V. / Rosenfeld, S.S. / Moores, C.A.
CitationJournal: J Biol Chem / Year: 2012
Title: The structural basis of force generation by the mitotic motor kinesin-5.
Authors: Adeline Goulet / William M Behnke-Parks / Charles V Sindelar / Jennifer Major / Steven S Rosenfeld / Carolyn A Moores /
Abstract: Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved ...Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor's properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.
History
DepositionApr 19, 2012Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 21, 2012Provider: repository / Type: Initial release
Revision 1.1Jan 16, 2013Group: Database references
Revision 1.2Apr 19, 2017Group: Other
Revision 1.3Aug 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 1.4May 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 ALPHA-1D CHAIN
B: TUBULIN BETA-2B CHAIN
C: KINESIN-LIKE PROTEIN KIF11
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,6627
Polymers141,8173
Non-polymers1,8454
Water00
1


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

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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-5 / KINESIN-LIKE PROTEIN 1 / KINESIN-LIKE SPINDLE PRO KINESIN-RELATED MOTOR PROTEIN EG5 / ...KINESIN-5 / KINESIN-LIKE PROTEIN 1 / KINESIN-LIKE SPINDLE PRO KINESIN-RELATED MOTOR PROTEIN EG5 / THYROID RECEPTOR-INTERACTING PROTEIN 5 / TR-INTERACTING PROTEIN 5 / TRIP-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) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 / References: UniProt: P52732

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Non-polymers , 4 types, 4 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-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: 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
#7: Chemical ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Details

Compound detailsENGINEERED RESIDUE IN CHAIN C, CYS 25 TO VAL ENGINEERED RESIDUE IN CHAIN C, CYS 43 TO SER ...ENGINEERED RESIDUE IN CHAIN C, CYS 25 TO VAL ENGINEERED RESIDUE IN CHAIN C, CYS 43 TO SER ENGINEERED RESIDUE IN CHAIN C, CYS 87 TO ALA ENGINEERED RESIDUE IN CHAIN C, CYS 99 TO ALA ENGINEERED RESIDUE IN CHAIN C, THR 126 TO CYS
Sequence detailsCYS-LIGHT CONSTRUCT (C25V,C43S,C87A,C99A). THE MUTATION T126C WAS INTRODUCED FOR GOLD-LABELLING.

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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: 13-PROTOFILAMENT MICROTUBULE-BOUND HUMAN KINESIN-5 MOTOR DOMAIN IN ABSENCE OF NUCLEOTIDES
Type: COMPLEX
Buffer solutionName: 80 MM PIPES, 5 MM MGCL2, 1 MM EGTA, 1 U/ML APYRASE / pH: 6.8 / Details: 80 MM PIPES, 5 MM MGCL2, 1 MM EGTA, 1 U/ML APYRASE
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 16, 2011
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: 2300 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: 138
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1Flex-EMmodel fitting
2UCSF Chimeramodel fitting
3FREALIGN3D reconstruction
4SPIDER3D reconstruction
CTF correctionDetails: FREALIGN
3D reconstructionResolution: 9.5 Å / Num. of particles: 6748 / Nominal pixel size: 2.8 Å
Details: THE LOOP 5 (119-131) WAS MODELED WITH MODELLER. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2078. (DEPOSITION ID: 10752).
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: METHOD--RIGID BODY (1JFF). RIGID BODY AND FLEXIBLE FITTING FOR THE CHIMERIC MODEL BUILT USING 1II6 AND 3HQD. REFINEMENT PROTOCOL--X-RAY
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11II6

1ii6

11II61PDBexperimental model
23HQD

3hqd

13HQD2PDBexperimental model
31JFF

1jff

11JFF3PDBexperimental model
RefinementHighest resolution: 9.5 Å
Refinement stepCycle: LAST / Highest resolution: 9.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1188 0 123 0 1311

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