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

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Basic information

Entry
Database: PDB / ID: 6mlr
TitleCryo-EM structure of microtubule-bound Kif7 in the AMPPNP state
Components
  • Kinesin-like protein KIF7
  • Tubulin alpha-1A chain
  • Tubulin beta chain
Keywordsmotor protein/inhibitor / Microtubule tip-tracking / Primary cilium / Hedgehog signaling / MOTOR PROTEIN / motor protein-inhibitor complex
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
Specimen sourceHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsMani, N. / Jiang, S. / Wilson-Kubalek, E.M. / Ku, P. / Milligan, R.A. / Subramanian, R.
Funding supportUnited States , 1件
OrganizationGrant numberCountry
R37GM052468United 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 27, 2018 / Release: May 1, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 1, 2019Structure modelrepositoryInitial release
1.1May 8, 2019Structure modelData collection / Database referencescitation / citation_author_citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-9141
  • Imaged by UCSF Chimera
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)146,0197
Polymers143,6923
Non-polymers2,3264
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein/peptide , 3 types, 3 molecules ABC

#1: Protein/peptide 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
#2: Protein/peptide 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
#3: Protein/peptide Kinesin-like protein KIF7


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

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Guanosine diphosphate / Comment: GDP (energy-carrying molecule) *YM
#6: Chemical ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Paclitaxel
#7: 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

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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

ComponentName: Microtubule-bound Kif7 / Type: COMPLEX / Entity ID: 1, 2, 3 / Source: RECOMBINANT
Source (natural)Organism: sus scrofa (pig)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 6.8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: not available
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 FIELDBright-field microscopy
Image recordingAverage 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 scansMovie frames/image: 40 / Used frames/image: 0-40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
7UCSF Chimera1.11.2model fitting
9PHENIX1.12-2829model refinement
13FREALIX3D reconstruction
Image processingDetails: The images were corrected by motionCorr2
CTF correctionDetails: CTF correction using CTFFIND4 / Type: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: -25.76 ° / Axial rise/subunit: 8.83 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 84000
Details: segmnets were picked along helical segments manually using appion
3D reconstructionResolution: 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 buildingProtocol: FLEXIBLE FIT
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-ID
13J6GA
23J6GB
34A14A
RefinementHighest resolution: 4.2 Å
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.0079380
f_angle_d1.10112746
f_dihedral_angle_d10.7125550
f_chiral_restr0.061418
f_plane_restr0.0071651

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