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- EMDB-2152: Electron cryo-microscopy of microtubule-bound human kinesin-5 mot... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2152 | |||||||||
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Title | Electron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in the AMPPNP state (gold cluster in the loop5 T126C). | |||||||||
![]() | 3D 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) | |||||||||
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![]() | electron cryo-microscopy / kinesin / microtubule / mitosis / cancer | |||||||||
Function / homology | Kinesin motor domain, conserved site / Alpha tubulin / Beta tubulin, autoregulation binding site![]() | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 12.0 Å | |||||||||
![]() | Goulet A / Behnke-Parks WM / Sindelar CV / Major J / Rosenfeld SS / Moores CA | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 252.5 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.7 KB 11.7 KB | Display Display | ![]() |
Images | ![]() | 168.4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2077C ![]() 2078C ![]() 2079C ![]() 2080C ![]() 2081C ![]() 4aqvC ![]() 4aqwC C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D 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) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : 13-protofilament microtubule-bound human kinesin-5 motor domain w...
Entire | Name: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the loop5 (T126C). |
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Components |
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-Supramolecule #1000: 13-protofilament microtubule-bound human kinesin-5 motor domain w...
Supramolecule | Name: 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
Macromolecule | Name: alpha tubulin / type: protein_or_peptide / ID: 1 / Name.synonym: TUBULIN ALPHA-1D CHAIN / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() |
Sequence | InterPro: Alpha tubulin |
-Macromolecule #2: beta tubulin
Macromolecule | Name: beta tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: TUBULIN BETA-2B CHAIN / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() |
Sequence | InterPro: Beta tubulin, autoregulation binding site |
-Macromolecule #3: Kinesin-5 motor domain
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | InterPro: Kinesin motor domain, conserved site |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 6.8 / Details: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5 mM AMPPNP |
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Grid | Details: 400 mesh holey carbon grids |
Vitrification | Cryogen 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
Microscope | FEI TECNAI F20 |
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Temperature | Average: 90 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification |
Date | May 15, 2012 |
Image recording | Category: 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 beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination 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 stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
Details | The particles were selected along individual microtubules. |
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CTF correction | Details: FREALIGN |
Final reconstruction | Resolution.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 |