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- EMDB-9140: Cryo-EM structure of microtubule-bound Kif7 in the ADP state -

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Basic information

Entry
Database: EMDB / ID: EMD-9140
TitleCryo-EM structure of microtubule-bound Kif7 in the ADP state
Map data
SampleMicrotubule-bound Kif7:
Tubulin alpha-1A chain / Tubulin beta chain / Kinesin-like protein KIF7 / (ligand) x 5
Function / homology
Function and homology information


Hedgehog 'on' state / Hedgehog 'off' state / ciliary tip / negative regulation of smoothened signaling pathway / positive regulation of smoothened signaling pathway / ciliary basal body / kinesin complex / microtubule motor activity / microtubule-based movement / microtubule-based process ...Hedgehog 'on' state / Hedgehog 'off' state / ciliary tip / negative regulation of smoothened signaling pathway / positive regulation of smoothened signaling pathway / ciliary basal body / kinesin complex / microtubule motor activity / microtubule-based movement / microtubule-based process / cilium / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / microtubule / microtubule binding / ATPase activity / GTPase activity / GTP binding / ATP binding / cytoplasm
Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin/FtsZ, GTPase domain superfamily / Kinesin-like protein / P-loop containing nucleoside triphosphate hydrolase / Tubulin, C-terminal / Kinesin motor domain, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, conserved site / Beta tubulin, autoregulation binding site ...Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin/FtsZ, GTPase domain superfamily / Kinesin-like protein / P-loop containing nucleoside triphosphate hydrolase / Tubulin, C-terminal / Kinesin motor domain, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, conserved site / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ family, GTPase domain / Beta tubulin / Alpha tubulin / Kinesin motor domain / Tubulin / Kinesin motor domain superfamily / Kinesin motor domain signature. / Kinesin motor domain / Tubulin C-terminal domain / Kinesin motor domain profile. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin-beta mRNA autoregulation signal.
Tubulin alpha-1A chain / Tubulin beta chain / Kinesin-like protein KIF7
SourceHomo sapiens (human) / Pig (pig)
Methodhelical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWilson-Kubalek EM / Jiang S / Mani N / Ku P / Milligan RA / Subramanian R
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.
Validation ReportPDB-ID: 6mlq

SummaryFull reportAbout validation report
DateDeposition: Sep 27, 2018 / Header (metadata) release: Nov 14, 2018 / Map release: May 1, 2019 / Update: May 8, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6mlq
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9140.map.gz / Format: CCP4 / Size: 2.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 86 pix.
= 112.66 Å
1.31 Å/pix.
x 128 pix.
= 167.68 Å
1.31 Å/pix.
x 61 pix.
= 79.91 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: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.017 / Movie #1: 0.017
Minimum - Maximum-0.064982116 - 0.1256204
Average (Standard dev.)0.0031752898 (±0.012358681)
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-0.0650.1260.003

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Supplemental data

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Sample components

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Entire Microtubule-bound Kif7

EntireName: Microtubule-bound Kif7 / Number of components: 9

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Component #1: protein, Microtubule-bound Kif7

ProteinName: Microtubule-bound Kif7 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)

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Component #2: protein, Tubulin alpha-1A chain

ProteinName: Tubulin alpha-1A chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 50.121266 kDa
SourceSpecies: Pig (pig)

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Component #3: protein, Tubulin beta chain

ProteinName: Tubulin beta chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 49.90777 kDa
SourceSpecies: Pig (pig)

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Component #4: protein, Kinesin-like protein KIF7

ProteinName: Kinesin-like protein KIF7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 43.663262 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, GUANOSINE-5'-TRIPHOSPHATE

LigandName: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.52318 kDa

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Component #7: ligand, GUANOSINE-5'-DIPHOSPHATE

LigandName: GUANOSINE-5'-DIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.443201 kDa

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Component #8: ligand, TAXOL

LigandName: TAXOLPaclitaxel / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.853906 kDa

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Component #9: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 11.11 Å / Delta phi: -23.84 %deg;
Sample solutionSpecimen conc.: 0.5 mg/mL / pH: 6.8
Support filmnot available
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 280 K / Humidity: 90 % / 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
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 37 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1059

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: BACK PROJECTION / Software: FREALIX / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible
Input PDB model: 3J6G, 3J6G, 4A14
Chain ID: A, B, C
Output model

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