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- PDB-6b0l: KLP10A-AMPPNP in complex with a microtubule -

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Basic information

Entry
Database: PDB / ID: 6b0l
TitleKLP10A-AMPPNP in complex with a microtubule
Components
  • Kinesin-like protein Klp10A
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsMOTOR PROTEIN/STRUCTURAL PROTEIN / kinesin 13 / microtubule / tubulin / depolymerization / MOTOR PROTEIN-STRUCTURAL PROTEIN complex
Function / homology
Function and homology information


establishment of mitotic spindle asymmetry / establishment of meiotic spindle orientation / cortical microtubule / plus-end specific microtubule depolymerization / asymmetric protein localization involved in cell fate determination / meiotic spindle pole / mitotic spindle astral microtubule / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / centriole assembly ...establishment of mitotic spindle asymmetry / establishment of meiotic spindle orientation / cortical microtubule / plus-end specific microtubule depolymerization / asymmetric protein localization involved in cell fate determination / meiotic spindle pole / mitotic spindle astral microtubule / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / centriole assembly / mitotic chromosome movement towards spindle pole / kinetochore microtubule / meiotic spindle organization / spindle assembly involved in female meiosis I / non-motile cilium assembly / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / plus-end-directed microtubule motor activity / meiotic spindle / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / microtubule depolymerization / COPI-mediated anterograde transport / microtubule motor activity / spindle organization / kinesin complex / microtubule-based movement / mitotic spindle pole / cytoskeletal motor activity / centrosome duplication / microtubule-based process / chromosome, centromeric region / mitotic spindle organization / structural constituent of cytoskeleton / microtubule cytoskeleton organization / spindle pole / spindle / microtubule cytoskeleton / mitotic cell cycle / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cell division / GTPase activity / centrosome / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Kinesin-like protein KIF2A-like, N-terminal / Kinesin-like protein / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain ...Kinesin-like protein KIF2A-like, N-terminal / Kinesin-like protein / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain / 60s Ribosomal Protein L30; Chain: A; / 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 / Helix Hairpins / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin beta chain / Tubulin beta chain / Tubulin alpha-1B chain / Kinesin-like protein Klp10A
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.98 Å
AuthorsBenoit, M.P.M.H. / Asenjo, A.B. / Sosa, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM113164 United States
CitationJournal: Nat Commun / Year: 2018
Title: Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s.
Authors: Matthieu P M H Benoit / Ana B Asenjo / Hernando Sosa /
Abstract: Kinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promote microtubule depolymerization and lack motile activity. The molecular mechanism by which kinesin- ...Kinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promote microtubule depolymerization and lack motile activity. The molecular mechanism by which kinesin-13s depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue, here we report the near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to curved or straight tubulin in different nucleotide states. These structures show how nucleotide induced conformational changes near the catalytic site are coupled with movement of the kinesin-13-specific loop-2 to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.
History
DepositionSep 14, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 2, 2018Provider: repository / Type: Initial release
Revision 1.1May 9, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta chain
K: Kinesin-like protein Klp10A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,5559
Polymers147,1803
Non-polymers2,3756
Water00
1
A: Tubulin alpha-1B chain
B: Tubulin beta chain
K: Kinesin-like protein Klp10A
hetero molecules
x 35


Theoretical massNumber of molelcules
Total (without water)5,234,431315
Polymers5,151,303105
Non-polymers83,128210
Water0
TypeNameSymmetry operationNumber
helical symmetry operation34
identity operation1_555x,y,z1
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 35 / Rise per n subunits: 5.5 Å / Rotation per n subunits: 168.083 °)

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Components

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Protein , 3 types, 3 molecules ABK

#1: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: Q2XVP4
#2: Protein Tubulin beta chain


Mass: 49999.887 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: F2Z5B2, UniProt: P02554*PLUS
#3: Protein Kinesin-like protein Klp10A / Kinesin-like protein at cytological position 10A


Mass: 46975.758 Da / Num. of mol.: 1 / Fragment: neck-motor (UNP residues 197-615)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Klp10A, CG1453 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q960Z0

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Non-polymers , 5 types, 6 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.: 2 / 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
#8: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1KLP10A-AMPPMP in complex with a microtubuleCOMPLEX#1-#30MULTIPLE SOURCES
2KLP10A-AMPPMPCOMPLEX#31RECOMBINANT
3microtubuleORGANELLE OR CELLULAR COMPONENT#1-#21NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Drosophila melanogaster (fruit fly)7227
23Sus scrofa (pig)9823
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 6.8
Buffer component
IDConc.NameBuffer-ID
140 mMK-PIPES1
220 uMPaclitaxel1
35 mMMagnesium chloride1
44 mMAMP-PNP1
51 mMEGTA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Calibrated magnification: 46598 X / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.28 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategoryDetails
2Leginonbetaimage acquisition
4Gctf0.5CTF correction
7RosettaEM2017.13.59376model fitting
8PHENIX1.13.2998model fitting
9MODELLERmodel fittingUsed through UCSF chimera
10UCSF Chimera1.10.2model fitting
11Coot0.8.6model fitting
14SPIDER22.1initial Euler assignment
15FREALIGN9.11final Euler assignment
17FREALIGN9.113D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 168.083 ° / Axial rise/subunit: 5.5 Å / Axial symmetry: C1
Particle selectionDetails: manual picking of filaments
3D reconstructionResolution: 3.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6040
Details: 2 independent reconstructions, each using a distinct half of every filament.
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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