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- EMDB-1701: The molecular mechanism of the multi-tasking kinesin-8 motor -

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Basic information

Entry
Database: EMDB / ID: EMD-1701
TitleThe molecular mechanism of the multi-tasking kinesin-8 motor
Map data10A reconstruction of a 15pf microtubule decorated with the motor domain of Kif18A (kinesin 8) in the Apo-state
Sample
  • Sample: Microtubule complexed with motor domain of Kif18A (no nucleotide state)
  • Protein or peptide: Motor domain of Kif18A
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: Beta tubulin
KeywordsMolecular motor / kinesin 8 / ruby-helix
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodhelical reconstruction / cryo EM / Resolution: 10.1 Å
AuthorsPeters C / Brejc K / Belmont L / Bodey A / Lee Y / Yu M / Ramchandani S / Guo J / Lichtsteiner S / Wood KW ...Peters C / Brejc K / Belmont L / Bodey A / Lee Y / Yu M / Ramchandani S / Guo J / Lichtsteiner S / Wood KW / Sakowicz R / Hartman J / Moores C
CitationJournal: EMBO J / Year: 2010
Title: Insight into the molecular mechanism of the multitasking kinesin-8 motor.
Authors: Carsten Peters / Katjuša Brejc / Lisa Belmont / Andrew J Bodey / Yan Lee / Ming Yu / Jun Guo / Roman Sakowicz / James Hartman / Carolyn A Moores /
Abstract: Members of the kinesin-8 motor class have the remarkable ability to both walk towards microtubule plus-ends and depolymerise these ends on arrival, thereby regulating microtubule length. To analyse ...Members of the kinesin-8 motor class have the remarkable ability to both walk towards microtubule plus-ends and depolymerise these ends on arrival, thereby regulating microtubule length. To analyse how kinesin-8 multitasks, we studied the structure and function of the kinesin-8 motor domain. We determined the first crystal structure of a kinesin-8 and used cryo-electron microscopy to calculate the structure of the microtubule-bound motor. Microtubule-bound kinesin-8 reveals a new conformation compared with the crystal structure, including a bent conformation of the α4 relay helix and ordering of functionally important loops. The kinesin-8 motor domain does not depolymerise stabilised microtubules with ATP but does form tubulin rings in the presence of a non-hydrolysable ATP analogue. This shows that, by collaborating, kinesin-8 motor domain molecules can release tubulin from microtubules, and that they have a similar mechanical effect on microtubule ends as kinesin-13, which enables depolymerisation. Our data reveal aspects of the molecular mechanism of kinesin-8 motors that contribute to their unique dual motile and depolymerising functions, which are adapted to control microtubule length.
History
DepositionFeb 9, 2010-
Header (metadata) releaseMar 10, 2010-
Map releaseOct 1, 2010-
UpdateSep 9, 2011-
Current statusSep 9, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

4db-apyrase_...

Downloads & links

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Map

FileDownload / File: emd_1701.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation10A reconstruction of a 15pf microtubule decorated with the motor domain of Kif18A (kinesin 8) in the Apo-state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 320 pix.
= 448. Å		1.4 Å/pix.
x 320 pix.
= 448. Å		1.4 Å/pix.
x 320 pix.
= 448. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-6.53806 - 8.76477
Average (Standard dev.)0.00000000675811 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-159-160-160
Dimensions320320320
Spacing320320320
CellA=B=C: 448 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z448.000448.000448.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ121121121
MAP C/R/S123
start NC/NR/NS-160-159-160
NC/NR/NS320320320
D min/max/mean-6.5388.7650.000

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Supplemental data

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Sample components

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Entire : Microtubule complexed with motor domain of Kif18A (no nucleotide ...

EntireName: Microtubule complexed with motor domain of Kif18A (no nucleotide state)
Components
  • Sample: Microtubule complexed with motor domain of Kif18A (no nucleotide state)
  • Protein or peptide: Motor domain of Kif18A
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: Beta tubulin

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Supramolecule #1000: Microtubule complexed with motor domain of Kif18A (no nucleotide ...

SupramoleculeName: Microtubule complexed with motor domain of Kif18A (no nucleotide state)
type: sample / ID: 1000 / Number unique components: 3

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Macromolecule #1: Motor domain of Kif18A

MacromoleculeName: Motor domain of Kif18A / type: protein_or_peptide / ID: 1 / Name.synonym: Motor domain of Kif18A / Details: Contains the N-terminal 355 aa / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Recombinant expressionOrganism: Escherichia coli Rosetta DE3

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Macromolecule #2: Alpha tubulin

MacromoleculeName: Alpha tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: Alpha tubulin / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / Tissue: brain / Location in cell: Cytoplasm

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Macromolecule #3: Beta tubulin

MacromoleculeName: Beta tubulin / type: protein_or_peptide / ID: 3 / Name.synonym: Beta tubulin / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / Tissue: Brain / Location in cell: Cytoplasm

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Details: 80mM PIPES, 150mM NaCl, 7mM MgCl2, 1mM EGTA, 1mM beta-mercaptoethanol
GridDetails: 400 mesh copper grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Manual plunger
Timed resolved state: Vitrified after incubating with Apyrase
Method: Blotted for 1 sec before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lense stigmatism was corrected at 150,000 times magnification
DetailsLow dose
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 50 / Average electron dose: 10 e/Å2 / Bits/pixel: 12
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.0 mm / Nominal defocus max: 3.3 µm / Nominal defocus min: 0.92 µm / Nominal magnification: 50000
Sample stageSpecimen holder: cryo-holder / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Ruby-Helix
Details: Fourier Bessel Synthesis using data from 25 microtubules (36000 asymmetric units)
CTF correctionDetails: Phase flipping, Wiener

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