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- EMDB-1038: Nucleotide-induced conformations in the neck region of dimeric ki... -

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Basic information

Entry
Database: EMDB / ID: EMD-1038
TitleNucleotide-induced conformations in the neck region of dimeric kinesin.
Map datarat kinesin Dimer with an SH3 domain cloned within neck region rKS379, complexed to microtubules in the presence of AMP-PNP
Sample
  • Sample: rat kinesin construct rK379
  • Protein or peptide: rat kinesin
  • Protein or peptide: tubulin
Biological speciesRattus norvegicus (Norway rat)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 25.0 Å
AuthorsSkiniotis G
CitationJournal: EMBO J / Year: 2003
Title: Nucleotide-induced conformations in the neck region of dimeric kinesin.
Authors: Georgios Skiniotis / Thomas Surrey / Stephan Altmann / Heinz Gross / Young-Hwa Song / Eckhard Mandelkow / Andreas Hoenger /
Abstract: The neck region of kinesin constitutes a key component in the enzyme's walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin ...The neck region of kinesin constitutes a key component in the enzyme's walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed to microtubules. To this end we enhanced the visibility of this region by engineering an SH3 domain into the transition between neck linker and neck coiled coil. The resulting chimeric kinesin constructs remained functional as verified by physiology assays. In the presence of AMP-PNP the SH3 domains allowed us to identify the position of the neck in a well defined conformation and revealed its high flexibility in the absence of nucleotide. We show here the double-headed binding of dimeric kinesin along the same protofilament, which is characterized by the opposite directionality of neck linkers. In this configuration the neck coiled coil appears fully zipped. The position of the neck region in dimeric constructs is not affected by the presence of the tubulin C-termini as confirmed by subtilisin treatment of microtubules prior to motor decoration.
History
DepositionFeb 28, 2003-
Header (metadata) releaseFeb 28, 2003-
Map releaseMar 28, 2003-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 57.274318921
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 57.274318921
  • 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_1038.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrat kinesin Dimer with an SH3 domain cloned within neck region rKS379, complexed to microtubules in the presence of AMP-PNP
Voxel sizeX=Y=Z: 5.526 Å
Density
Contour Level1: 64.700000000000003 / Movie #1: 57.2743189
Minimum - Maximum0.0 - 100.0
Average (Standard dev.)43.457099999999997 (±9.864089999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 552.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.5265.5265.526
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z552.600552.600552.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean0.000100.00043.457

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Supplemental data

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Sample components

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Entire : rat kinesin construct rK379

EntireName: rat kinesin construct rK379
Components
  • Sample: rat kinesin construct rK379
  • Protein or peptide: rat kinesin
  • Protein or peptide: tubulin

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Supramolecule #1000: rat kinesin construct rK379

SupramoleculeName: rat kinesin construct rK379 / type: sample / ID: 1000 / Oligomeric state: dimer / Number unique components: 2

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Macromolecule #1: rat kinesin

MacromoleculeName: rat kinesin / type: protein_or_peptide / ID: 1 / Name.synonym: molecular motor / Number of copies: 1 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: rat kinesin
Molecular weightExperimental: 980 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET3

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Macromolecule #2: tubulin

MacromoleculeName: tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: microtubule / Number of copies: 1 / Oligomeric state: hetero-dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: rat kinesin / Tissue: brain / Cell: neuronal cells / Location in cell: cytoplasm
Molecular weightExperimental: 110 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 6.8
Details: PIPES 80 mM, MgCl2 1 mM, GTP 1 mM, Taxol 20 uM, DMSO 7.5%
StainingType: NEGATIVE / Details: ice-embeded
GridDetails: holey grids
VitrificationCryogen name: ETHANE / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: self made

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG/ST
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 38000
Sample stageSpecimen holder: side-entry / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 95 K
Alignment procedureLegacy - Astigmatism: was corrected at 180,000 times mag.
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 21 µm / Number real images: 16 / Average electron dose: 5 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PHOELIX, SUPREME
Details: Final map from 33 averaged datasets = 17 helical tubes

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