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- PDB-5nd2: Microtubule-bound MKLP2 motor domain in the presence of ADP -

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Basic information

Entry
Database: PDB / ID: 5nd2
TitleMicrotubule-bound MKLP2 motor domain in the presence of ADP
Components
  • Kinesin-like protein KIF20AKIF20A
  • Tubulin alpha chain
  • Tubulin beta-2B chain
KeywordsMOTOR PROTEIN / Kinesin Mitosis Microtubules
Function / homology
Function and homology information


Mitotic Telophase/Cytokinesis / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation ...Mitotic Telophase/Cytokinesis / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / COPI-dependent Golgi-to-ER retrograde traffic / RHO GTPases activate IQGAPs / COPI-independent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases Activate Formins / Kinesins / MHC class II antigen presentation / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Aggrephagy / COPI-dependent Golgi-to-ER retrograde traffic / The role of GTSE1 in G2/M progression after G2 checkpoint / Separation of Sister Chromatids / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / Recruitment of NuMA to mitotic centrosomes / midbody abscission / Regulation of PLK1 Activity at G2/M Transition / Hedgehog 'off' state / MHC class II antigen presentation / microtubule bundle formation / positive regulation of axon guidance / kinesin complex / microtubule motor activity / intercellular bridge / microtubule-based movement / mitotic cytokinesis / cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / regulation of cytokinesis / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / spindle / microtubule cytoskeleton organization / microtubule cytoskeleton / protein transport / double-stranded RNA binding / mitotic cell cycle / nervous system development / midbody / microtubule binding / microtubule / hydrolase activity / protein heterodimerization activity / GTPase activity / ubiquitin protein ligase binding / GTP binding / protein kinase binding / Golgi apparatus / ATP hydrolysis activity / nucleoplasm / ATP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
: / 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 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin ...: / 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 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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 / Tubulin alpha chain / Tubulin alpha-1B chain / Kinesin-like protein KIF20A / Tubulin beta-2B chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsAtherton, J. / Yu, I.M. / Cook, A. / Muretta, J.M. / Joseph, A.P. / Major, J. / Sourigues, Y. / Clause, J. / Topf, M. / Rosenfeld, S.S. ...Atherton, J. / Yu, I.M. / Cook, A. / Muretta, J.M. / Joseph, A.P. / Major, J. / Sourigues, Y. / Clause, J. / Topf, M. / Rosenfeld, S.S. / Houdusse, A. / Moores, C.A.
Funding support United Kingdom, 13items
OrganizationGrant numberCountry
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
United Kingdom
CitationJournal: Elife / Year: 2017
Title: The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.
Authors: Joseph Atherton / I-Mei Yu / Alexander Cook / Joseph M Muretta / Agnel Joseph / Jennifer Major / Yannick Sourigues / Jeffrey Clause / Maya Topf / Steven S Rosenfeld / Anne Houdusse / Carolyn A Moores /
Abstract: MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% ...MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.
History
DepositionMar 7, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 4, 2017Provider: repository / Type: Initial release

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Assembly

Deposited unit
C: Kinesin-like protein KIF20A
A: Tubulin alpha chain
B: Tubulin beta-2B chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,3329
Polymers156,0363
Non-polymers2,2966
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9240 Å2
ΔGint-68 kcal/mol
Surface area42930 Å2
MethodPISA
Number of models5

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Components

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Protein , 3 types, 3 molecules CAB

#1: Protein Kinesin-like protein KIF20A / KIF20A / Kinesin-like protein 174 / Rab6-interacting kinesin-like protein / Rabkinesin-6


Mass: 56021.332 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: MKLP2 25-520 with bound ADP / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Kif20a, Rab6kifl / Production host: Escherichia coli (E. coli) / References: UniProt: P97329
#2: Protein Tubulin alpha chain


Mass: 50107.238 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Alpha-tubulin / Source: (natural) Bos taurus (cattle) / Plasmid details: Cytoskeleton Inc / References: UniProt: F2Z4C1, UniProt: P81947*PLUS
#3: Protein Tubulin beta-2B chain


Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Plasmid details: Cytoskeleton Inc / References: UniProt: Q6B856

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Non-polymers , 5 types, 6 molecules

#4: 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
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: 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
#7: 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
#8: Chemical ChemComp-TA1 / TAXOL / Paclitaxel


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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of 14pf microtubule with bound MKLP2 motor domain in the presence of ADPCOMPLEXMicrotubule bound to taxol and GMPCPP#1-#30MULTIPLE SOURCES
2Kinesin-like protein KIF20AKIF20ACOMPLEXMicrotubule bound to taxol and GMPCPP#11RECOMBINANT
3Tubulin alpha and beta chainsCOMPLEXMicrotubule bound to taxol and GMPCPP#2-#31NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Bos taurus (cattle)9913
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9842
Details: Gold-standard FSCtrue using noise substitution test (Chen et al., 2014)
Symmetry type: POINT

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