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- PDB-6mlq: Cryo-EM structure of microtubule-bound Kif7 in the ADP state -

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Basic information

Entry
Database: PDB / ID: 6mlq
TitleCryo-EM structure of microtubule-bound Kif7 in the ADP state
Components
  • Kinesin-like protein KIF7
  • Tubulin alpha-1A chain
  • Tubulin beta chain
KeywordsMOTOR PROTEIN / Microtubule tip-tracking / Primary cilium / Hedgehog signaling
Function / homology
Function and homology information


ciliary tip / positive regulation of smoothened signaling pathway / kinesin complex / microtubule motor activity / motile cilium / microtubule-based movement / Hedgehog 'off' state / negative regulation of smoothened signaling pathway / Hedgehog 'on' state / structural constituent of cytoskeleton ...ciliary tip / positive regulation of smoothened signaling pathway / kinesin complex / microtubule motor activity / motile cilium / microtubule-based movement / Hedgehog 'off' state / negative regulation of smoothened signaling pathway / Hedgehog 'on' state / structural constituent of cytoskeleton / microtubule cytoskeleton organization / fibrillar center / neuron migration / mitotic cell cycle / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / cilium / ciliary basal body / intracellular membrane-bounded organelle / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Kinesin-like protein / 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. ...Kinesin-like protein / 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 / Tubulin alpha-1A chain / Tubulin beta chain / Kinesin-like protein KIF7
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsMani, N. / Jiang, S. / Wilson-Kubalek, E.M. / Ku, P. / Milligan, R.A. / Subramanian, R.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Dev Cell / Year: 2019
Title: Interplay between the Kinesin and Tubulin Mechanochemical Cycles Underlies Microtubule Tip Tracking by the Non-motile Ciliary Kinesin Kif7.
Authors: Shuo Jiang / Nandini Mani / Elizabeth M Wilson-Kubalek / Pei-I Ku / Ronald A Milligan / Radhika Subramanian /
Abstract: The correct localization of Hedgehog effectors to the tip of primary cilia is critical for proper signal transduction. The conserved non-motile kinesin Kif7 defines a "cilium-tip compartment" by ...The correct localization of Hedgehog effectors to the tip of primary cilia is critical for proper signal transduction. The conserved non-motile kinesin Kif7 defines a "cilium-tip compartment" by localizing to the distal ends of axonemal microtubules. How Kif7 recognizes microtubule ends remains unknown. We find that Kif7 preferentially binds GTP-tubulin at microtubule ends over GDP-tubulin in the mature microtubule lattice, and ATP hydrolysis by Kif7 enhances this discrimination. Cryo-electron microscopy (cryo-EM) structures suggest that a rotated microtubule footprint and conformational changes in the ATP-binding pocket underlie Kif7's atypical microtubule-binding properties. Finally, Kif7 not only recognizes but also stabilizes a GTP-form of tubulin to promote its own microtubule-end localization. Thus, unlike the characteristic microtubule-regulated ATPase activity of kinesins, Kif7 modulates the tubulin mechanochemical cycle. We propose that the ubiquitous kinesin fold has been repurposed in Kif7 to facilitate organization of a spatially restricted platform for localization of Hedgehog effectors at the cilium tip.
History
DepositionSep 27, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2019Provider: repository / Type: Initial release
Revision 1.1May 8, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Jun 12, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.4Mar 13, 2024Group: Data collection / Database references / 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
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

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Structure visualization

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  • Deposited structure unit
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  • EMDB-9140
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Tubulin alpha-1A chain
B: Tubulin beta chain
C: Kinesin-like protein KIF7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,9889
Polymers143,6923
Non-polymers2,2966
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Tubulin alpha-1A chain / Alpha-tubulin 1 / Tubulin alpha-1 chain


Mass: 50121.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02550
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554
#3: Protein Kinesin-like protein KIF7


Mass: 43663.262 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KIF7, UNQ340/PRO539 / Plasmid: pET-28a(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q2M1P5

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Non-polymers , 5 types, 6 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


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


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*YM
#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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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Microtubule-bound Kif7 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3) / Plasmid: pET 28a(+)
Buffer solutionpH: 6.8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: not available
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 280 K / Details: Blotted from behind the grid for 2 seconds

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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
Image recordingAverage exposure time: 8 sec. / Electron dose: 37 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1059
Image scansMovie frames/image: 40 / Used frames/image: 0-40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
7UCSF Chimera1.11.2model fitting
9PHENIX1.12-2829model refinement
13FREALIX3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: -23.84 ° / Axial rise/subunit: 11.11 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 84000
Details: Segments were picked along helical segments manually using appion
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15919 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
13J6G

3j6g

A3J6G1
23J6G

3j6g

B3J6G1
34A14

4a14

C4A142
RefinementHighest resolution: 4.2 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059383
ELECTRON MICROSCOPYf_angle_d1.03412752
ELECTRON MICROSCOPYf_dihedral_angle_d6.3735556
ELECTRON MICROSCOPYf_chiral_restr0.0611419
ELECTRON MICROSCOPYf_plane_restr0.0081654

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