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- EMDB-1340: The beginning of kinesin's force-generating cycle visualized at 9... -

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Basic information

Entry
Database: EMDB / ID: EMD-1340
TitleThe beginning of kinesin's force-generating cycle visualized at 9-A resolution.
Map data9-Angstrom map of nucleotide-free kinesin complexed to the microtubule
Sample
  • Sample: Nucleotide-free kinesin bound to a 13-protofilament microtubule
  • Protein or peptide: Kinesin
  • Protein or peptide: Alpha-tubulin
  • Protein or peptide: Beta-tubulin
Function / homology
Function and homology information


regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / anterograde neuronal dense core vesicle transport / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / ciliary rootlet ...regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / anterograde neuronal dense core vesicle transport / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / ciliary rootlet / lysosome localization / positive regulation of potassium ion transport / plus-end-directed microtubule motor activity / vesicle transport along microtubule / RHO GTPases activate KTN1 / Kinesins / positive regulation of axon guidance / kinesin complex / microtubule motor activity / centrosome localization / mitochondrion transport along microtubule / COPI-dependent Golgi-to-ER retrograde traffic / motile cilium / microtubule-based movement / stress granule disassembly / natural killer cell mediated cytotoxicity / Insulin processing / synaptic vesicle transport / postsynaptic cytosol / microtubule-based process / phagocytic vesicle / axon cytoplasm / MHC class II antigen presentation / dendrite cytoplasm / axon guidance / positive regulation of synaptic transmission, GABAergic / regulation of membrane potential / positive regulation of protein localization to plasma membrane / cellular response to type II interferon / structural constituent of cytoskeleton / microtubule cytoskeleton organization / centriolar satellite / neuron migration / Signaling by ALK fusions and activated point mutants / mitotic cell cycle / microtubule cytoskeleton / nuclear membrane / microtubule binding / vesicle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / cadherin binding / protein heterodimerization activity / GTPase activity / GTP binding / protein-containing complex binding / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / metal ion binding / identical protein binding / membrane / cytoplasm / cytosol
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 / Kinesin-1 heavy chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsSindelar CV / Downing KH
CitationJournal: J Cell Biol / Year: 2007
Title: The beginning of kinesin's force-generating cycle visualized at 9-A resolution.
Authors: Charles V Sindelar / Kenneth H Downing /
Abstract: We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, ...We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in "ejecting" adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the "neck linker"). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself.
History
DepositionMar 12, 2007-
Header (metadata) releaseMar 13, 2007-
Map releaseJun 7, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2p4n
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2p4n
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2p4n
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1340.gif

Downloads & links

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Map

FileDownload / File: emd_1340.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation9-Angstrom map of nucleotide-free kinesin complexed to the microtubule
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 180 pix.
= 360. Å		2 Å/pix.
x 180 pix.
= 360. Å		2 Å/pix.
x 180 pix.
= 360. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.0766 / Movie #1: 0.065
Minimum - Maximum-0.0903918 - 0.164345
Average (Standard dev.)0.00348294 (±0.0324056)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-90-90-90
Dimensions180180180
Spacing180180180
CellA=B=C: 360 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-90-90-90
NC/NR/NS180180180
D min/max/mean-0.0900.1640.003

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Supplemental data

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Sample components

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Entire : Nucleotide-free kinesin bound to a 13-protofilament microtubule

EntireName: Nucleotide-free kinesin bound to a 13-protofilament microtubule
Components
  • Sample: Nucleotide-free kinesin bound to a 13-protofilament microtubule
  • Protein or peptide: Kinesin
  • Protein or peptide: Alpha-tubulin
  • Protein or peptide: Beta-tubulin

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Supramolecule #1000: Nucleotide-free kinesin bound to a 13-protofilament microtubule

SupramoleculeName: Nucleotide-free kinesin bound to a 13-protofilament microtubule
type: sample / ID: 1000
Oligomeric state: The 13-protofilament microtubule forms a pseudo helix interrupted by a single seam
Number unique components: 3

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Macromolecule #1: Kinesin

MacromoleculeName: Kinesin / type: protein_or_peptide / ID: 1 / Name.synonym: K349
Details: C220 "cys-lite" K349 construct: 6 of 8 native cysteines eliminated, introduced cysteine at position 220
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 36.4134 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: BL21

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Macromolecule #2: Alpha-tubulin

MacromoleculeName: Alpha-tubulin / type: protein_or_peptide / ID: 2 / Details: Glycerol-free tubulin purchased from Cytoskeleton / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Brain / Location in cell: Brain
Molecular weightExperimental: 50.1279 KDa

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Macromolecule #3: Beta-tubulin

MacromoleculeName: Beta-tubulin / type: protein_or_peptide / ID: 3 / Details: Glycerol-free tubulin purchased from Cytoskeleton / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Brain
Molecular weightExperimental: 49.9283 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8 / Details: 25mM Pipes, 25mM NaCl, 2mM MgCl2, 1mM EGTA
GridDetails: 300 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Home-built
Method: Grids were not glow discharged. Liquid was almost entirely wicked away prior to blotting. Blotting time was less than 0.5 sec.

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Electron microscopy

MicroscopeJEOL 4000EX
TemperatureAverage: 105 K
Alignment procedureLegacy - Astigmatism: e.g objective lens astigmatism was corrected at 400,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6.3 µm / Number real images: 350 / Average electron dose: 16 e/Å2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 400 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.1 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: CTF correction was integrated into the back-projection process
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Data from 188 microtubules were incorporated into the final reconstruction
Number images used: 150000

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Atomic model buiding 1

Initial model(PDB ID:

1jff

,

1bg2

)
SoftwareName: SITUS
DetailsProtocol: Rigid body. Kinesin and tubulin were separately fitted into the final map using the program SITUS.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-2p4n:
Human Monomeric Kinesin (1BG2) and Bovine Tubulin (1JFF) Docked into the 9-Angstrom Cryo-EM Map of Nucleotide-Free Kinesin Complexed to the Microtubule

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