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- EMDB-1025: Microscopic evidence for a minus-end-directed power stroke in the... -

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Basic information

Entry
Database: EMDB / ID: EMD-1025
TitleMicroscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd.
Map datahelical reconstruction of SH3 tagged Ncd296 motor fragment no nucleotide state
Sample
  • Sample: SH3 tagged Ncd296 motor fragment from Drosophila Melanogaster no nucleotide state
  • Protein or peptide: SH3 tagged Ncd296 dimer
  • Protein or peptide: tubulin dimer
Biological speciesDrosophila melanogaster (fruit fly) / Bos taurus (domestic cattle)
Methodhelical reconstruction / cryo EM / negative staining
AuthorsWendt TG
CitationJournal: EMBO J / Year: 2002
Title: Microscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd.
Authors: Thomas G Wendt / Niels Volkmann / Georgios Skiniotis / Kenneth N Goldie / Jens Müller / Eckhard Mandelkow / Andreas Hoenger /
Abstract: We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various ...We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various nucleotide conditions, and applied molecular docking using ncd's dimeric X-ray structure to generate a mechanistic model for force transduction. To visualize the alpha-helical coiled-coil neck better, we engineered an SH3 domain to the N-terminal end of our ncd construct (296-700). Ncd exhibits strikingly different nucleotide-dependent three-dimensional conformations and microtubule-binding patterns from those of conventional kinesin. In the absence of nucleotide, the neck adapts a configuration close to that found in the X-ray structure with stable interactions between the neck and motor core domain. Minus-end-directed movement is based mainly on two key events: (i) the stable neck-core interactions in ncd generate a binding geometry between motor and microtubule which places the motor ahead of its cargo in the minus-end direction; and (ii) after the uptake of ATP, the two heads rearrange their position relative to each other in a way that promotes a swing of the neck in the minus-end direction.
History
DepositionFeb 26, 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: 18
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 18
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1025.gif

Downloads & links

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Map

FileDownload / File: emd_1025.map.gz / Format: CCP4 / Size: 2.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhelical reconstruction of SH3 tagged Ncd296 motor fragment no nucleotide state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.53 Å/pix.
x 73 pix.
= 403.69 Å		5.53 Å/pix.
x 101 pix.
= 558.53 Å		5.53 Å/pix.
x 101 pix.
= 558.53 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: 5.53 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 14.0 / Movie #1: 18
Minimum - Maximum-38.0 - 46.0
Average (Standard dev.)2.52698 (±11.1553)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-36
Dimensions10110173
Spacing10110173
CellA: 558.53 Å / B: 558.53 Å / C: 403.69 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.535.535.53
M x/y/z10110173
origin x/y/z0.0000.0000.000
length x/y/z558.530558.530403.690
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-50-50-36
NC/NR/NS10110173
D min/max/mean-38.00046.0002.527

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Supplemental data

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Sample components

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Entire : SH3 tagged Ncd296 motor fragment from Drosophila Melanogaster no ...

EntireName: SH3 tagged Ncd296 motor fragment from Drosophila Melanogaster no nucleotide state
Components
  • Sample: SH3 tagged Ncd296 motor fragment from Drosophila Melanogaster no nucleotide state
  • Protein or peptide: SH3 tagged Ncd296 dimer
  • Protein or peptide: tubulin dimer

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Supramolecule #1000: SH3 tagged Ncd296 motor fragment from Drosophila Melanogaster no ...

SupramoleculeName: SH3 tagged Ncd296 motor fragment from Drosophila Melanogaster no nucleotide state
type: sample / ID: 1000 / Oligomeric state: SH3ncd forms a dimer / Number unique components: 2

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Macromolecule #1: SH3 tagged Ncd296 dimer

MacromoleculeName: SH3 tagged Ncd296 dimer / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: SH3ncd296 forms a dimer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightExperimental: 106.8 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: tubulin dimer

MacromoleculeName: tubulin dimer / type: protein_or_peptide / ID: 2 / Name.synonym: alpha beta tubulin
Details: bovine tubulin was purchased from Cytoskeleton Inc. (Denver,CO)
Oligomeric state: polymer of alpha beta dimers / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightExperimental: 100 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

Concentration2.45 mg/mL
BufferpH: 6.8 / Details: 80mM PIPES, 2mM MgCl2
StainingType: NEGATIVE / Details: plunge-frozen in liquid ethane
GridDetails: quantifoil holey grids
VitrificationCryogen name: ETHANE / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: standard guillotine. vitrification carried out at room temperature

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG/ST
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 150K mag
Detailslow dose imaging
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 21 µm / Number real images: 14 / Od range: 0.9 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 160 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.26 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 38000
Sample stageSpecimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Details257 subunits per 137 helical turns
Final reconstructionApplied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Software - Name: PHOELIX
Details: final map was calculated from 27 datasets of near and far sides

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