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- EMDB-9141: Cryo-EM structure of microtubule-bound Kif7 in the AMPPNP state -

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Basic information

Entry
Database: EMDB / ID: EMD-9141
TitleCryo-EM structure of microtubule-bound Kif7 in the AMPPNP state
Map dataKif7 bound to microtubules in the AMPPNP state
Sample
  • Complex: Microtubule-bound Kif7
    • Protein or peptide: Tubulin alpha-1A chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Kinesin-like protein KIF7
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOLPaclitaxel
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsMicrotubule tip-tracking / Primary cilium / Hedgehog signaling / MOTOR PROTEIN / motor protein-inhibitor complex
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 speciessus scrofa (pig) / Sus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWilson-Kubalek EM / Jiang S / Mani N / Ku P / Milligan RA / Subramanian R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R37GM052468 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, 2018-
Header (metadata) releaseNov 7, 2018-
Map releaseMay 1, 2019-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0147
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0147
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mlr
  • Surface level: 0.0147
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9141.png

Downloads & links

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Map

FileDownload / File: emd_9141.map.gz / Format: CCP4 / Size: 2.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKif7 bound to microtubules in the AMPPNP state
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.0147 / Movie #1: 0.0147
Minimum - Maximum-0.07830961 - 0.14318249
Average (Standard dev.)0.0020820154 (±0.018225778)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin37-444
Dimensions1286186
Spacing6112886
CellA: 79.909996 Å / B: 167.68 Å / C: 112.659996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z6112886
origin x/y/z0.0000.0000.000
length x/y/z79.910167.680112.660
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-43744
NC/NR/NS6112886
D min/max/mean-0.0780.1430.002

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Supplemental data

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Sample components

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Entire : Microtubule-bound Kif7

EntireName: Microtubule-bound Kif7
Components
  • Complex: Microtubule-bound Kif7
    • Protein or peptide: Tubulin alpha-1A chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Kinesin-like protein KIF7
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOLPaclitaxel
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Microtubule-bound Kif7

SupramoleculeName: Microtubule-bound Kif7 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: sus scrofa (pig)

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Macromolecule #1: Tubulin alpha-1A chain

MacromoleculeName: Tubulin alpha-1A chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (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

UniProtKB: Tubulin alpha-1A chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (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

UniProtKB: Tubulin beta chain

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Macromolecule #3: Kinesin-like protein KIF7

MacromoleculeName: Kinesin-like protein KIF7 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.663262 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 TAS

UniProtKB: Kinesin-like protein KIF7

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / 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 #5: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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Macromolecule #6: TAXOL

MacromoleculeName: TAXOL / type: ligand / ID: 6 / Number of copies: 1 / Formula: TA1
Molecular weightTheoretical: 853.906 Da
Chemical component information

ChemComp-TA1:
TAXOL / medication, chemotherapy*YM / Paclitaxel

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Macromolecule #7: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 7 / 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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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
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: not available
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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 0-40 / Number grids imaged: 1 / Number real images: 1059 / Average exposure time: 8.0 sec. / Average electron dose: 37.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 84000
Details: segmnets were picked along helical segments manually using appion
Startup modelType of model: OTHER
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 8.83 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.76 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIX / Number images used: 37220
DetailsThe images were corrected by motionCorr2

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Atomic model buiding 1

Initial model
PDB IDChain

3j6g

chain_id: A, source_name: PDB, initial_model_type: experimental model

3j6g

chain_id: B, source_name: PDB, initial_model_type: experimental model

4a14

chain_id: A, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6mlr:
Cryo-EM structure of microtubule-bound Kif7 in the AMPPNP state

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