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Yorodumi- EMDB-2077: Electron cryo-microscopy of microtubule-bound human kinesin-5 mot... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2077 | |||||||||
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Title | Electron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in AMPPNP state. | |||||||||
Map data | 3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site | |||||||||
Sample |
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Keywords | cancer / electron cryo-microscopy / kinesin / microtubule / mitosis | |||||||||
Function / homology | Function and homology information spindle elongation / regulation of mitotic centrosome separation / Kinesins / plus-end-directed microtubule motor activity / mitotic centrosome separation / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / microtubule motor activity / kinesin complex / spindle organization ...spindle elongation / regulation of mitotic centrosome separation / Kinesins / plus-end-directed microtubule motor activity / mitotic centrosome separation / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / microtubule motor activity / kinesin complex / spindle organization / microtubule-based movement / microtubule-based process / mitotic spindle assembly / MHC class II antigen presentation / mitotic spindle organization / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / mitotic spindle / spindle pole / microtubule cytoskeleton organization / spindle / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule binding / microtubule / hydrolase activity / protein heterodimerization activity / cell division / GTPase activity / GTP binding / protein kinase binding / protein-containing complex / ATP binding / membrane / nucleus / metal ion binding / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | Bos taurus (cattle) / Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 9.7 Å | |||||||||
Authors | Goulet A / Behnke-Parks WM / Sindelar C / Rosenfeld S / Moores C | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2077.map.gz | 238.1 KB | EMDB map data format | |
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Header (meta data) | emd-2077-v30.xml emd-2077.xml | 12.3 KB 12.3 KB | Display Display | EMDB header |
Images | emd_2077.jpg | 144.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2077 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2077 | HTTPS FTP |
-Validation report
Summary document | emd_2077_validation.pdf.gz | 254.7 KB | Display | EMDB validaton report |
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Full document | emd_2077_full_validation.pdf.gz | 253.9 KB | Display | |
Data in XML | emd_2077_validation.xml.gz | 4.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2077 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2077 | HTTPS FTP |
-Related structure data
Related structure data | 4aqvMC 2078C 2079C 2080C 2081C 2152C 4aqwC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2077.map.gz / Format: CCP4 / Size: 348.6 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
-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 |
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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 type: sample / ID: 1000 Oligomeric state: 13-protofilament microtubule with one kineisn-5 motor domain bound every tubulin heterodimers Number unique components: 2 |
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-Macromolecule #1: alpha-beta tubulin dimer
Macromolecule | Name: alpha-beta tubulin dimer / type: protein_or_peptide / ID: 1 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: brain |
-Macromolecule #2: Kinesin-5 motor domain
Macromolecule | Name: Kinesin-5 motor domain / type: protein_or_peptide / ID: 2 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pet21a |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 6.8 / Details: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5mM 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 |
-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 | Jan 10, 2011 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 46 / Average electron dose: 18 e/Å2 / Bits/pixel: 8 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-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: 9.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN Details: Approximately 50,000 asymmetric units were averaged in the final reconstruction. Number images used: 3587 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera, FlexEM |
Details | Protocol: rigid body then flexible fitting. The domain was fitted as a rigid body. The N-terminal residues 6 to 16 were built in the EM map and the final model was refined by flexible fitting. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation |
Output model | PDB-4aqv: |
-Atomic model buiding 2
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B |
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Software | Name: Chimera |
Details | Protocol: rigid body. alpha- and b-tubulin were separately fitted. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation |
Output model | PDB-4aqv: |