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- EMDB-1428: A lever-arm rotation drives motility of the minus-end-directed ki... -

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Basic information

Entry
Database: EMDB / ID: EMD-1428
TitleA lever-arm rotation drives motility of the minus-end-directed kinesin Ncd.
Map dataNcd in its ATP bound, AMPPNP, state on 15 filament microtubules
Sample
  • Sample: non-claret disjunction atp, amppnp, state
  • Protein or peptide: tubulin
  • Protein or peptide: ncd
Biological speciesBos taurus (cattle)
Methodhelical reconstruction / cryo EM / Resolution: 18.0 Å
AuthorsEndres NF / Yoshioka C / Milligan RA / Vale RD
CitationJournal: Nature / Year: 2006
Title: A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd.
Authors: Nicholas F Endres / Craig Yoshioka / Ronald A Milligan / Ronald D Vale /
Abstract: Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that ...Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described. By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors (such as Drosophila Ncd) are less well understood. Using cryo-electron microscopy, here we demonstrate that a coiled-coil mechanical element of microtubule-bound Ncd rotates approximately 70 degrees towards the minus end upon ATP binding. Extending or shortening this coiled coil increases or decreases velocity, respectively, without affecting ATPase activity. An unusual Ncd mutant that lacks directional preference shows unstable nucleotide-dependent conformations of its coiled coil, underscoring the role of this mechanical element in motility. These results show that the force-producing conformational change in Ncd occurs on ATP binding, as in other kinesins, but involves the swing of a lever-arm mechanical element similar to that described for myosins.
History
DepositionSep 19, 2007-
Header (metadata) releaseSep 19, 2007-
Map releaseSep 19, 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: 6.54
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 6.54
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1428.gif

Downloads & links

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Map

FileDownload / File: emd_1428.map.gz / Format: CCP4 / Size: 2.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNcd in its ATP bound, AMPPNP, state on 15 filament microtubules
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
5 Å/pix.
x 73 pix.
= 365. Å		5 Å/pix.
x 101 pix.
= 505. Å		5 Å/pix.
x 101 pix.
= 505. 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: 5 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 29.699999999999999 / Movie #1: 6.54
Minimum - Maximum-33.148899999999998 - 45.8508
Average (Standard dev.)2.31237 (±12.287100000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions10110173
Spacing10110173
CellA: 505 Å / B: 505 Å / C: 365 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z555
M x/y/z10110173
origin x/y/z0.0000.0000.000
length x/y/z505.000505.000365.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ10110173
MAP C/R/S213
start NC/NR/NS000
NC/NR/NS10110173
D min/max/mean-33.14945.8512.312

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Supplemental data

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Sample components

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Entire : non-claret disjunction atp, amppnp, state

EntireName: non-claret disjunction atp, amppnp, state
Components
  • Sample: non-claret disjunction atp, amppnp, state
  • Protein or peptide: tubulin
  • Protein or peptide: ncd

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Supramolecule #1000: non-claret disjunction atp, amppnp, state

SupramoleculeName: non-claret disjunction atp, amppnp, state / type: sample / ID: 1000
Oligomeric state: Motors bound to 15 protofilament helical microtubules
Number unique components: 2

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Macromolecule #1: tubulin

MacromoleculeName: tubulin / type: protein_or_peptide / ID: 1 / Oligomeric state: helical 15 protofilaments / Recombinant expression: Yes
Source (natural)Organism: Bos taurus (cattle) / synonym: Cow / Tissue: brain
Recombinant expressionOrganism: Bos taurus (cattle)

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Macromolecule #2: ncd

MacromoleculeName: ncd / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Bos taurus (cattle) / synonym: Cow
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET-17b plasmid Invitrogen

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration10 mg/mL
BufferpH: 7.4
Details: 25 mM MOPS (pH 7.25), 100 mM NaCl, 2 mM MgCl2, 1 mM EGTA and 1 mM dithiothreitol, 5mg/ml tubulin, 5mM AMPPNP
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 4 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: 3 sec blot

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Average electron dose: 10 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal magnification: 39000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: phoelix

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