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- EMDB-1035: A new look at the microtubule binding patterns of dimeric kinesins. -

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Basic information

Entry
Database: EMDB / ID: EMD-1035
TitleA new look at the microtubule binding patterns of dimeric kinesins.
Map dataNeurospora crassa kinesin monomer (nK343) AMP-PNP state
Sample
  • Sample: neurospora crassa kinesin monomer
  • Protein or peptide: neurospora kinesin
  • Protein or peptide: tubulin
Biological speciesNeurospora crassa (fungus)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 25.0 Å
AuthorsHoenger A
CitationJournal: J Mol Biol / Year: 2000
Title: A new look at the microtubule binding patterns of dimeric kinesins.
Authors: A Hoenger / M Thormählen / R Diaz-Avalos / M Doerhoefer / K N Goldie / J Müller / E Mandelkow /
Abstract: The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good ...The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.
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: 51.666842714
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 51.666842714
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1035.gif

Downloads & links

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Map

FileDownload / File: emd_1035.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNeurospora crassa kinesin monomer (nK343) AMP-PNP state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.68 Å/pix.
x 100 pix.
= 567.6 Å		5.68 Å/pix.
x 100 pix.
= 567.6 Å		5.68 Å/pix.
x 100 pix.
= 567.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.676 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 56.200000000000003 / Movie #1: 51.6668427
Minimum - Maximum0.0 - 100.0
Average (Standard dev.)36.123699999999999 (±9.0769)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 567.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.6765.6765.676
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z567.600567.600567.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean0.000100.00036.124

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Supplemental data

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Sample components

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Entire : neurospora crassa kinesin monomer

EntireName: neurospora crassa kinesin monomer
Components
  • Sample: neurospora crassa kinesin monomer
  • Protein or peptide: neurospora kinesin
  • Protein or peptide: tubulin

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Supramolecule #1000: neurospora crassa kinesin monomer

SupramoleculeName: neurospora crassa kinesin monomer / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 2

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Macromolecule #1: neurospora kinesin

MacromoleculeName: neurospora kinesin / type: protein_or_peptide / ID: 1 / Name.synonym: molecular motor / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Neurospora crassa (fungus) / synonym: neurospora kinesin

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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: No / Database: NCBI
Source (natural)Organism: Neurospora crassa (fungus) / synonym: neurospora kinesin / Tissue: brain / Cell: neuronal cells / Location in cell: cytoplasm

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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: 6.9
Details: Pipes 20mM, 50 mM NaCl, 5mM mgcl,1mM Mg-ATP, 20um taxol.
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 CM120T
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 21 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: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 37000
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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