+Open data
-Basic information
Entry | Database: PDB / ID: 6mlr | ||||||
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Title | Cryo-EM structure of microtubule-bound Kif7 in the AMPPNP state | ||||||
Components |
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Keywords | motor protein/inhibitor / Microtubule 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 / Hedgehog 'off' state / microtubule motor activity / kinesin complex / microtubule-based movement / negative regulation of smoothened signaling pathway / ciliary basal body / Hedgehog 'on' state / cilium ...ciliary tip / positive regulation of smoothened signaling pathway / Hedgehog 'off' state / microtubule motor activity / kinesin complex / microtubule-based movement / negative regulation of smoothened signaling pathway / ciliary basal body / Hedgehog 'on' state / cilium / structural constituent of cytoskeleton / microtubule cytoskeleton organization / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / mitotic cell cycle / microtubule binding / microtubule / hydrolase activity / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | Homo sapiens (human) Sus scrofa (pig) | ||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å | ||||||
Authors | Mani, N. / Jiang, S. / Wilson-Kubalek, E.M. / Ku, P. / Milligan, R.A. / Subramanian, R. | ||||||
Funding support | United States, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6mlr.cif.gz | 223.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6mlr.ent.gz | 171.9 KB | Display | PDB format |
PDBx/mmJSON format | 6mlr.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6mlr_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 6mlr_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 6mlr_validation.xml.gz | 39.8 KB | Display | |
Data in CIF | 6mlr_validation.cif.gz | 58.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ml/6mlr ftp://data.pdbj.org/pub/pdb/validation_reports/ml/6mlr | HTTPS FTP |
-Related structure data
Related structure data | 9141MC 9140C 6mlqC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 3 types, 3 molecules ABC
#1: Protein | Mass: 50121.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02550 |
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#2: Protein | 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 | 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 (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q2M1P5 |
-Non-polymers , 4 types, 4 molecules
#4: Chemical | ChemComp-GTP / |
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#5: Chemical | ChemComp-GDP / |
#6: Chemical | ChemComp-TA1 / |
#7: Chemical | ChemComp-ANP / |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: Microtubule-bound Kif7 / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Source (natural) | Organism: sus scrofa (pig) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 6.8 |
Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: not available |
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 280 K / Details: Blotted from behind the grid for 2 seconds |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Average 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 scans | Movie frames/image: 40 / Used frames/image: 0-40 |
-Processing
Software | Name: PHENIX / Version: 1.12_2829: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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Image processing | Details: The images were corrected by motionCorr2 | ||||||||||||||||||||||||
CTF correction | Details: CTF correction using CTFFIND4 / Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: -25.76 ° / Axial rise/subunit: 8.83 Å / Axial symmetry: C1 | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 84000 Details: segmnets were picked along helical segments manually using appion | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37220 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: HELICAL | ||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT | ||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
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Refinement | Highest resolution: 4.2 Å | ||||||||||||||||||||||||
Refine LS restraints |
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