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- EMDB-24721: Complex of AMPPNP-Kif7 and Gli2 Zinc-Finger domain bound to micro... -

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Basic information

Entry
Database: EMDB / ID: EMD-24721
TitleComplex of AMPPNP-Kif7 and Gli2 Zinc-Finger domain bound to microtubules
Map dataComplex of Kif7_AMPPNP and Gli2 Zinc-finger domain bound to microtubules
Sample
  • Complex: Microtubule-bound complex of Kif7 and Gli2 zinc finger domain
    • Complex: Kinesin Kif7
      • Protein or peptide: Kinesin-like protein KIF7
    • Complex: Zinc-finger protein Gli2Zinc finger
      • Protein or peptide: Zinc finger protein GLI2
    • Complex: MicrotubulesMicrotubule
      • Protein or peptide: Tubulin alpha-1A chain
      • Protein or peptide: Tubulin beta chain
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


ciliary tip / positive regulation of smoothened signaling pathway / kinesin complex / microtubule motor activity / microtubule-based movement / Hedgehog 'off' state / negative regulation of smoothened signaling pathway / ciliary basal body / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Hedgehog 'on' state ...ciliary tip / positive regulation of smoothened signaling pathway / kinesin complex / microtubule motor activity / microtubule-based movement / Hedgehog 'off' state / negative regulation of smoothened signaling pathway / ciliary basal body / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Hedgehog 'on' state / cilium / structural constituent of cytoskeleton / 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
Tubulin alpha-1A chain / Tubulin beta chain / Kinesin-like protein KIF7
Similarity search - Component
Biological speciesHomo sapiens (human) / Sus scrofa (pig) / Pig (pig)
Methodhelical reconstruction / cryo EM / Resolution: 3.89 Å
AuthorsWilson-Kubalek EM / Mani N / Haque F / Freniere C / Milligan RA / Subramanian R
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateJul 27, 2022-
Current statusJul 27, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24721.png

Downloads & links

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Map

FileDownload / File: emd_24721.map.gz / Format: CCP4 / Size: 6.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComplex of Kif7_AMPPNP and Gli2 Zinc-finger domain bound to microtubules
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 101 pix.
= 131.3 Å		1.3 Å/pix.
x 130 pix.
= 169. Å		1.3 Å/pix.
x 121 pix.
= 157.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.031489246 - 0.06969108
Average (Standard dev.)0.0019961398 (±0.009818419)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions130121101
Spacing121130101
CellA: 157.29999 Å / B: 169.0 Å / C: 131.29999 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Microtubule-bound complex of Kif7 and Gli2 zinc finger domain

EntireName: Microtubule-bound complex of Kif7 and Gli2 zinc finger domain
Components
  • Complex: Microtubule-bound complex of Kif7 and Gli2 zinc finger domain
    • Complex: Kinesin Kif7
      • Protein or peptide: Kinesin-like protein KIF7
    • Complex: Zinc-finger protein Gli2Zinc finger
      • Protein or peptide: Zinc finger protein GLI2
    • Complex: MicrotubulesMicrotubule
      • Protein or peptide: Tubulin alpha-1A chain
      • Protein or peptide: Tubulin beta chain
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Microtubule-bound complex of Kif7 and Gli2 zinc finger domain

SupramoleculeName: Microtubule-bound complex of Kif7 and Gli2 zinc finger domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: Kinesin Kif7

SupramoleculeName: Kinesin Kif7 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Supramolecule #3: Zinc-finger protein Gli2

SupramoleculeName: Zinc-finger protein Gli2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)

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Supramolecule #4: Microtubules

SupramoleculeName: Microtubules / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Sus scrofa (pig)

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Macromolecule #1: Kinesin-like protein KIF7

MacromoleculeName: Kinesin-like protein KIF7 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 59.525902 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMGLEAQRLP GAEEAPVRVA LRVRPLLPKE LLHGHQSCLQ VEPGLGRVTL GRDRHFGFHV VLAEDAGQEA VYQACVQPLL EAFFEGFNA TVFAYGQTGS GKTYTMGEAS VASLLEDEQG IVPRAMAEAF KLIDENDLLD CLVHVSYLEV YKEEFRDLLE V GTASRDIQ ...String:
GMGLEAQRLP GAEEAPVRVA LRVRPLLPKE LLHGHQSCLQ VEPGLGRVTL GRDRHFGFHV VLAEDAGQEA VYQACVQPLL EAFFEGFNA TVFAYGQTGS GKTYTMGEAS VASLLEDEQG IVPRAMAEAF KLIDENDLLD CLVHVSYLEV YKEEFRDLLE V GTASRDIQ LREDERGNVV LCGVKEVDVE GLDEVLSLLE MGNAARHTGA THLNHLSSRS HTVFTVTLEQ RGRAPSRLPR PA PGQLLVS KFHFVDLAGS ERVLKTGSTG ERLKESIQIN SSLLALGNVI SALGDPQRRG SHIPYRDSKI TRILKDSLGG NAK TVMIAC VSPSSSDFDE TLNTLNYASR AQNIRNRATV NWRPEAERPP EETASGARGP PRHRSETRII HRGRRAPGPA TASA AAAMR LGAECARYRA CTDAAYSLLR ELQAEPGLPG AAARKVRDWL CAVEGERSAL SSASGPDSGI ESASVEDQAA QGAGG RKED EGAQQLLTLQ NQVARLEEEN RDFLAALEDA MEQYKLQSDR LREQQEEMVE LRLRLELVRP

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Macromolecule #2: Zinc finger protein GLI2

MacromoleculeName: Zinc finger protein GLI2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.847665 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: Tubulin alpha-1A chain

MacromoleculeName: Tubulin alpha-1A chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig)
Molecular weightTheoretical: 50.121266 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLIGQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRAHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YEPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

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Macromolecule #4: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig)
Molecular weightTheoretical: 49.90777 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEGEEDEA

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Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #6: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #7: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 6.8
Component:
ConcentrationFormulaName
80.0 mMC8H18N2O6S2PIPES
1.0 mMMgCl2Magnesium Chloride
2.0 mMEGTA
1.0 mMAMPPNP
GridModel: C-flat-1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 280 K / Instrument: HOMEMADE PLUNGER / Details: Blotted from behind the grid for 2 seconds.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 0-40 / Average exposure time: 9.0 sec. / Average electron dose: 36.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 50639
Details: Segments were picked along helical segments manually using Appion
CTF correctionSoftware - Name: CTFFIND (ver. 4) / Details: CTF correction using CTFFIND4
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.956 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.747 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.89 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIX / Number images used: 25730
DetailsThe images were corrected by motionCorr2

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Atomic model buiding 1

Initial model
PDB IDChain

3j6e

chain_id: A

3j6e

chain_id: B

6mlr

chain_id: C

2gli

chain_id: G
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 150 / Target criteria: Correlation coefficient
Output model

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

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