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- EMDB-5916: Cryo-Electron Microscopy of Nucleotide-free Kinesin motor domain ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5916
TitleCryo-Electron Microscopy of Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule
Map data3D structure of Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule
Sample
  • Sample: Cryo-Electron Microscopy of Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule
  • Protein or peptide: TUBULIN ALPHA CHAIN
  • Protein or peptide: TUBULIN BETA CHAIN
  • Protein or peptide: Kinesin heavy chain isoform 5C motor domain
KeywordsKIF5C / nucleotide-free kinesin / Rigor-conformation / GMPCPP-MT / Axonal transport / transport protein
Function / homology
Function and homology information


distal axon / anterograde dendritic transport of messenger ribonucleoprotein complex / anterograde axonal protein transport / apolipoprotein receptor binding / intracellular mRNA localization / ciliary rootlet / motor neuron axon guidance / kinesin complex / microtubule motor activity / motile cilium ...distal axon / anterograde dendritic transport of messenger ribonucleoprotein complex / anterograde axonal protein transport / apolipoprotein receptor binding / intracellular mRNA localization / ciliary rootlet / motor neuron axon guidance / kinesin complex / microtubule motor activity / motile cilium / mRNA transport / postsynaptic cytosol / axonal growth cone / axon cytoplasm / dendrite cytoplasm / GABA-ergic synapse / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / neuron projection / hydrolase activity / neuronal cell body / 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 / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. ...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 / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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 heavy chain isoform 5C
Similarity search - Component
Biological speciesSus scrofa (pig) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.1 Å
AuthorsMorikawa M / Yajima H / Nitta R / Inoue S / Ogura T / Sato C / Hirokawa N
CitationJournal: EMBO J / Year: 2015
Title: X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding.
Authors: Manatsu Morikawa / Hiroaki Yajima / Ryo Nitta / Shigeyuki Inoue / Toshihiko Ogura / Chikara Sato / Nobutaka Hirokawa /
Abstract: The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state ...The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the 'rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.
History
DepositionMar 3, 2014-
Header (metadata) releaseApr 16, 2014-
Map releaseApr 1, 2015-
UpdateJun 3, 2015-
Current statusJun 3, 2015Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 152
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 152
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j6h
  • Surface level: 152
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5916.tif

Downloads & links

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Map

FileDownload / File: emd_5916.map.gz / Format: CCP4 / Size: 199.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D structure of Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.5 Å/pix.
x 43 pix.
= 107.5 Å		2.5 Å/pix.
x 43 pix.
= 107.5 Å		2.5 Å/pix.
x 28 pix.
= 70. 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: 2.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 152.0 / Movie #1: 152
Minimum - Maximum-30.7155056 - 274.338867189999974
Average (Standard dev.)113.464569089999998 (±46.7421875)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-15-13-21
Dimensions432843
Spacing432843
CellA: 70.0 Å / B: 107.5 Å / C: 107.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z284343
origin x/y/z0.0000.0000.000
length x/y/z70.000107.500107.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-13-15-21
NC/NR/NS284343
D min/max/mean-30.716274.339113.465

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Supplemental data

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Sample components

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Entire : Cryo-Electron Microscopy of Nucleotide-free Kinesin motor domain ...

EntireName: Cryo-Electron Microscopy of Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule
Components
  • Sample: Cryo-Electron Microscopy of Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule
  • Protein or peptide: TUBULIN ALPHA CHAIN
  • Protein or peptide: TUBULIN BETA CHAIN
  • Protein or peptide: Kinesin heavy chain isoform 5C motor domain

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Supramolecule #1000: Cryo-Electron Microscopy of Nucleotide-free Kinesin motor domain ...

SupramoleculeName: Cryo-Electron Microscopy of Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule
type: sample / ID: 1000
Oligomeric state: One kineisn motor domain binds to one tubulin dimers
Number unique components: 3

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Macromolecule #1: TUBULIN ALPHA CHAIN

MacromoleculeName: TUBULIN ALPHA CHAIN / type: protein_or_peptide / ID: 1 / Name.synonym: alpha tubulin / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / Tissue: Brain
SequenceUniProtKB: Tubulin alpha-1A chain

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Macromolecule #2: TUBULIN BETA CHAIN

MacromoleculeName: TUBULIN BETA CHAIN / type: protein_or_peptide / ID: 2 / Name.synonym: beta tubulin / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / Tissue: Brain
SequenceUniProtKB: Tubulin beta chain

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Macromolecule #3: Kinesin heavy chain isoform 5C motor domain

MacromoleculeName: Kinesin heavy chain isoform 5C motor domain / type: protein_or_peptide / ID: 3 / Name.synonym: KIF5C motor domain / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET21b
SequenceUniProtKB: Kinesin heavy chain isoform 5C

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8 / Details: 100mM PIPES, 1mM EGTA, 1mM MgCl2
StainingType: NEGATIVE / Details: No staining
GridDetails: 300 mesh copper grid with thin carbon support, glow discharged in amylamine atmosphere
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: LEICA KF80 / Method: On grid blotting (Microtubule, KIF5C)

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Electron microscopy

MicroscopeJEOL 2010F
DateAug 18, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Average electron dose: 7 e/Å2
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe particles were selected along individual microtubules using an automatic processing program.
CTF correctionDetails: Each filament
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: OTHER / Software - Name: MATLAB, ImagicV / Number images used: 302000
Final two d classificationNumber classes: 18

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Atomic model buiding 1

Initial modelPDB ID:

1jff


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera, Modeller
DetailsInitial local fitting was done using Chimera, and for some loops Modeller was used.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Target criteria: Cross-correlation, Average map value, Atoms inside the contour
Output model

PDB-3j6h:
Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule

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Atomic model buiding 2

Initial modelPDB ID:

3wrd


Chain - Chain ID: K
SoftwareName: Chimera, Modeller
DetailsInitial local fitting was done using Chimera, and for some loops Modeller was used.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Target criteria: Cross-correlation, Average map value, Atoms inside the contour
Output model

PDB-3j6h:
Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule

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