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- EMDB-2538: cryo-electron 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-2538 | |||||||||
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Title | cryo-electron microscopy of microtubule-bound human kinesin-5 motor domain in the ADP state with a gold cluster attached to the loop5 (T126C) | |||||||||
![]() | Reconstruction of microtubule-bound human kinesin-5 motor domain in presence of ADP and with a glod cluster covalently attached to the residue T126C | |||||||||
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![]() | cryo-electron microscopy / kinesins / microtubules / mitosis / mechanochemistry | |||||||||
Function / homology | ![]() 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 / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 18.0 Å | |||||||||
![]() | GOULET A / MAJOR J / JUN Y / GROSS SP / ROSENFELD SR / MOORES CA | |||||||||
![]() | ![]() Title: Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family. Authors: Adeline Goulet / Jennifer Major / Yonggun Jun / Steven P Gross / Steven S Rosenfeld / Carolyn A Moores / ![]() Abstract: Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and ...Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin--the transport motor kinesin-1--is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor's structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements. | |||||||||
History |
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Structure visualization
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 455.1 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 11 KB 11 KB | Display Display | ![]() |
Images | ![]() | 150 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2533C ![]() 2534C ![]() 2535C ![]() 2536C ![]() 2537C ![]() 2539C ![]() 2540C ![]() 2541C ![]() 2542C ![]() 4ck5C ![]() 4ck6C ![]() 4ck7C C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Reconstruction of microtubule-bound human kinesin-5 motor domain in presence of ADP and with a glod cluster covalently attached to the residue T126C | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.2 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 i...
Entire | Name: 13-protofilament microtubule-bound human kinesin-5 motor domain in presence of ADP |
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Components |
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-Supramolecule #1000: 13-protofilament microtubule-bound human kinesin-5 motor domain i...
Supramolecule | Name: 13-protofilament microtubule-bound human kinesin-5 motor domain in presence of ADP type: sample / ID: 1000 Details: A gold cluster is covalently attached to the loop5 T126C 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 / Recombinant expression: No |
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Source (natural) | Organism: ![]() ![]() |
Sequence | UniProtKB: Tubulin alpha-1D chain / InterPro: Alpha tubulin |
-Macromolecule #2: beta tubulin
Macromolecule | Name: beta tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: TUBULIN BETA-2B CHAIN / Recombinant expression: No |
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Source (natural) | Organism: ![]() ![]() |
Sequence | UniProtKB: Tubulin beta-2B chain / 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: cys-lite mutant containing the substitution T126C. A undecagold cluster is covalently attached to T126C. Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Kinesin-like protein KIF11 / 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: 20 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 10 mM ADP, 2% glycerol |
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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 3.5 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 | Jan 16, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 20 / Average electron dose: 18 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 68000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.1 µm |
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 | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: SPIDER, FREALIGN Details: Approximately 135,000 asymmetric units were averaged in the final reconstruction. Number images used: 1000 |