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- EMDB-1028: Image reconstructions of microtubules decorated with monomeric an... -

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Basic information

Entry
Database: EMDB / ID: EMD-1028
TitleImage reconstructions of microtubules decorated with monomeric and dimeric kinesins: comparison with x-ray structure and implications for motility.
Map datarat kinesin dimer rK379 nucleotide-free state
Sample
  • Sample: Rat kinesin motor domain complexed to microtubules without nucleotides
  • Protein or peptide: rat kinesin
  • Protein or peptide: tubulin
Biological speciesRattus norvegicus (Norway rat)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 25.0 Å
AuthorsHoenger A
CitationJournal: J Cell Biol / Year: 1998
Title: Image reconstructions of microtubules decorated with monomeric and dimeric kinesins: comparison with x-ray structure and implications for motility.
Authors: A Hoenger / S Sack / M Thormählen / A Marx / J Müller / H Gross / E Mandelkow /
Abstract: We have decorated microtubules with monomeric and dimeric kinesin constructs, studied their structure by cryoelectron microscopy and three-dimensional image reconstruction, and compared the results ...We have decorated microtubules with monomeric and dimeric kinesin constructs, studied their structure by cryoelectron microscopy and three-dimensional image reconstruction, and compared the results with the x-ray crystal structure of monomeric and dimeric kinesin. A monomeric kinesin construct (rK354, containing only a short neck helix insufficient for coiled-coil formation) decorates microtubules with a stoichiometry of one kinesin head per tubulin subunit (alpha-beta-heterodimer). The orientation of the kinesin head (an anterograde motor) on the microtubule surface is similar to that of ncd (a retrograde motor). A longer kinesin construct (rK379) forms a dimer because of the longer neck helix forming a coiled-coil. Unexpectedly, this construct also decorates the microtubule with a stoichiometry of one head per tubulin subunit, and the orientation is similar to that of the monomeric construct. This means that the interaction with microtubules causes the two heads of a kinesin dimer to separate sufficiently so that they can bind to two different tubulin subunits. This result is in contrast to recent models and can be explained by assuming that the tubulin-kinesin interaction is antagonistic to the coiled-coil interaction within a kinesin dimer.
History
DepositionFeb 27, 2003-
Header (metadata) releaseFeb 27, 2003-
Map releaseFeb 27, 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: 61.479930355
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 61.479930355
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1028.gif

Downloads & links

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Map

FileDownload / File: emd_1028.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrat kinesin dimer rK379 nucleotide-free state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.71 Å/pix.
x 100 pix.
= 571.4 Å		5.71 Å/pix.
x 100 pix.
= 571.4 Å		5.71 Å/pix.
x 100 pix.
= 571.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.714 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 66.299999999999997 / Movie #1: 61.4799304
Minimum - Maximum0.0 - 100.0
Average (Standard dev.)45.539299999999997 (±9.70031)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 571.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.7145.7145.714
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z571.400571.400571.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean0.000100.00045.539

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Supplemental data

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Sample components

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Entire : Rat kinesin motor domain complexed to microtubules without nucleotides

EntireName: Rat kinesin motor domain complexed to microtubules without nucleotides
Components
  • Sample: Rat kinesin motor domain complexed to microtubules without nucleotides
  • Protein or peptide: rat kinesin
  • Protein or peptide: tubulin

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Supramolecule #1000: Rat kinesin motor domain complexed to microtubules without nucleotides

SupramoleculeName: Rat kinesin motor domain complexed to microtubules without nucleotides
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
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: tubulin

MacromoleculeName: tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: microtubules / Number of copies: 1 / Oligomeric state: hetero dimer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: rat kinesin / Tissue: brain / Cell: neuronal cells / Location in cell: cytoplasm
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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 80mM, MgCl 1mM, GTP 1mM, Taxol 20uM, DMSO 5%
StainingType: NEGATIVE / Details: ice-embedded
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 CM12
TemperatureAverage: 95 K
Image recordingCategory: FILM / Film or detector model: AGFA SCIENTA FILM / Digitization - Scanner: EMIL 10 / Digitization - Sampling interval: 20 µm / Number real images: 10 / Average electron dose: 5 e/Å2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 100 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 35000
Sample stageSpecimen holder: side-entry / Specimen holder model: GATAN LIQUID NITROGEN

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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, Suprim
Details: Final maps from 20 averaged datasets = 10 helical tubes

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