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- EMDB-2152: Electron cryo-microscopy of microtubule-bound human kinesin-5 mot... -

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Basic information

Entry
Database: EMDB / ID: EMD-2152
TitleElectron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in the AMPPNP state (gold cluster in the loop5 T126C).
Map data3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site in presence of AMPPNP (gold cluster attached in loop5, T126C)
Sample
  • Sample: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the loop5 (T126C).
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Kinesin-5 motor domain
Keywordselectron cryo-microscopy / kinesin / microtubule / mitosis / cancer
Function / homologyKinesin motor domain, conserved site / Alpha tubulin / Beta tubulin, autoregulation binding site
Function and homology information
Biological speciesBos taurus (cattle) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsGoulet A / Behnke-Parks WM / Sindelar CV / Major J / Rosenfeld SS / Moores CA
CitationJournal: J Biol Chem / Year: 2012
Title: The structural basis of force generation by the mitotic motor kinesin-5.
Authors: Adeline Goulet / William M Behnke-Parks / Charles V Sindelar / Jennifer Major / Steven S Rosenfeld / Carolyn A Moores /
Abstract: Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved ...Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor's properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.
History
DepositionJun 29, 2012-
Header (metadata) releaseJul 9, 2012-
Map releaseNov 21, 2012-
UpdateJan 9, 2013-
Current statusJan 9, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2152.jpg

Downloads & links

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Map

FileDownload / File: emd_2152.map.gz / Format: CCP4 / Size: 348.6 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site in presence of AMPPNP (gold cluster attached in loop5, T126C)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 45 pix.
= 126. Å		2.8 Å/pix.
x 45 pix.
= 126. Å		2.8 Å/pix.
x 45 pix.
= 126. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-6.44548321 - 7.18690872
Average (Standard dev.)0.17872882 (±1.9942255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-13-67-37
Dimensions454545
Spacing454545
CellA=B=C: 126.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z454545
origin x/y/z0.0000.0000.000
length x/y/z126.000126.000126.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-32-32-32
NX/NY/NZ646464
MAP C/R/S123
start NC/NR/NS-67-13-37
NC/NR/NS454545
D min/max/mean-6.4457.1870.179

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Supplemental data

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Sample components

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Entire : 13-protofilament microtubule-bound human kinesin-5 motor domain w...

EntireName: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the loop5 (T126C).
Components
  • Sample: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the loop5 (T126C).
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Kinesin-5 motor domain

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Supramolecule #1000: 13-protofilament microtubule-bound human kinesin-5 motor domain w...

SupramoleculeName: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the loop5 (T126C).
type: sample / ID: 1000
Oligomeric state: 13-protofilament microtubule with one kinesin-5 motor domain bound every tubulin heterodimers
Number unique components: 3

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Macromolecule #1: alpha tubulin

MacromoleculeName: alpha tubulin / type: protein_or_peptide / ID: 1 / Name.synonym: TUBULIN ALPHA-1D CHAIN / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: brain
SequenceInterPro: Alpha tubulin

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Macromolecule #2: beta tubulin

MacromoleculeName: beta tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: TUBULIN BETA-2B CHAIN / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: brain
SequenceInterPro: Beta tubulin, autoregulation binding site

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Macromolecule #3: Kinesin-5 motor domain

MacromoleculeName: Kinesin-5 motor domain / type: protein_or_peptide / ID: 3 / Name.synonym: KINESIN-LIKE PROTEIN KIF11
Details: undecagold cluster was attached to the specific cysteine residue in the loop5 (T126C)
Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21a
SequenceInterPro: Kinesin motor domain, conserved site

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8 / Details: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5 mM AMPPNP
GridDetails: 400 mesh holey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: chamber at 24 degrees C, blot 2.5 sec

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateMay 15, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 23 / Average electron dose: 18 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected along individual microtubules.
CTF correctionDetails: FREALIGN
Final reconstructionResolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: Approximately 17,000 asymmetric units were averaged in the final reconstruction. The deposited map is low pass filtered at 16 A
Number images used: 1302

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