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- PDB-7rx0: Complex of AMPPNP-Kif7 and Gli2 Zinc-Finger domain bound to micro... -

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Basic information

Entry
Database: PDB / ID: 7rx0
TitleComplex of AMPPNP-Kif7 and Gli2 Zinc-Finger domain bound to microtubules
Components
  • Kinesin-like protein KIF7
  • Tubulin alpha-1A chain
  • Tubulin beta chain
  • Zinc finger protein GLI2
KeywordsMOTOR PROTEIN / Kinesin / Microtubule / Transcription factor / motor domain
Function / homology
Function and homology information


ciliary tip / spindle elongation / positive regulation of smoothened signaling pathway / microtubule motor activity / kinesin complex / microtubule-based movement / Hedgehog 'off' state / negative regulation of smoothened signaling pathway / mitotic spindle organization / ciliary basal body ...ciliary tip / spindle elongation / positive regulation of smoothened signaling pathway / microtubule motor activity / kinesin complex / microtubule-based movement / Hedgehog 'off' state / negative regulation of smoothened signaling pathway / mitotic spindle organization / ciliary basal body / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Hedgehog 'on' state / structural constituent of cytoskeleton / cilium / microtubule cytoskeleton organization / mitotic cell cycle / microtubule binding / microtubule / hydrolase activity / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / 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 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin ...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 / 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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / 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: 3.89 Å
AuthorsMani, N. / Wilson-Kubalek, E.M. / Haque, F. / Freniere, C. / Milligan, R.A. / Subramanian, R.
Funding support United States, 1items
OrganizationGrant numberCountry
American Cancer Society United States
CitationJournal: Nat Cell Biol / Year: 2022
Title: Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.
Authors: Farah Haque / Christian Freniere / Qiong Ye / Nandini Mani / Elizabeth M Wilson-Kubalek / Pei-I Ku / Ronald A Milligan / Radhika Subramanian /
Abstract: A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI ...A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear. Here, we uncover DNA mimicry by the ciliary kinesin KIF7 as a mechanism for the recruitment of GLI to microtubules, wherein the coiled-coil dimerization domain of KIF7, characterized by its striking shape, size and charge similarity to DNA, forms a complex with the DNA-binding zinc fingers in GLI, thus revealing a mode of tethering a DNA-binding protein to the cytoskeleton. GLI increases KIF7 microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive GLI tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We retooled this coiled-coil-based GLI-KIF7 interaction to inhibit the nuclear and cilium localization of GLI. This strategy can potentially be exploited to downregulate erroneously activated GLI in human cancers.
History
DepositionAug 20, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.name
Revision 1.2Jul 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3Jul 27, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Kinesin-like protein KIF7
G: Zinc finger protein GLI2
A: Tubulin alpha-1A chain
B: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)168,9778
Polymers167,4034
Non-polymers1,5754
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9220 Å2
ΔGint-37 kcal/mol
Surface area51170 Å2

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Components

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Protein , 4 types, 4 molecules CGAB

#1: Protein Kinesin-like protein KIF7


Mass: 59525.902 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KIF7, UNQ340/PRO539 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q2M1P5
#2: Protein Zinc finger protein GLI2


Mass: 7847.665 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
#3: 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
#4: 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

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Non-polymers , 4 types, 4 molecules

#5: 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
#6: 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
#7: Chemical ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-CPP, energy-carrying molecule analogue*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Microtubule-bound complex of Kif7 and Gli2 zinc finger domainCOMPLEX#1-#40MULTIPLE SOURCES
2Kinesin Kif7COMPLEX#11RECOMBINANT
3Zinc-finger protein Gli2COMPLEX#21RECOMBINANT
4MicrotubulesCOMPLEX#3-#41NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Sus scrofa (pig)9823
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)
Buffer solutionpH: 6.8
Buffer component
IDConc.NameFormulaBuffer-ID
180 mMPIPESC8H18N2O6S21
21 mMMagnesium ChlorideMgCl21
32 mMEGTA1
41 mMAMPPNP1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-1.2/1.3
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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 36 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40 / Used frames/image: 0-40

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
7UCSF Chimera1.14model fitting
12FREALIX3D reconstruction
19PHENIX1.17.1-3660model refinement
Image processingDetails: The images were corrected by motionCorr2
CTF correctionDetails: CTF correction using CTFFIND4 / Type: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: -25.747 ° / Axial rise/subunit: 8.956 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 50639
Details: Segments were picked along helical segments manually using Appion
3D reconstructionResolution: 3.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25730 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Atomic model buildingB value: 150 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building

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

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
13J6E

3j6e

A3J6E1
23J6E

3j6e

B3J6E1
36MLR

6mlr

C6MLR2
42GLI

2gli

G2GLI3
RefinementHighest resolution: 3.89 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059866
ELECTRON MICROSCOPYf_angle_d0.64413408
ELECTRON MICROSCOPYf_dihedral_angle_d11.3441380
ELECTRON MICROSCOPYf_chiral_restr0.0421505
ELECTRON MICROSCOPYf_plane_restr0.0041750

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