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- EMDB-1340: The beginning of kinesin's force-generating cycle visualized at 9... -
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Basic information
Entry | Database: EMDB / ID: EMD-1340 | |||||||||
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Title | The beginning of kinesin's force-generating cycle visualized at 9-A resolution. | |||||||||
![]() | 9-Angstrom map of nucleotide-free kinesin complexed to the microtubule | |||||||||
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Function / homology | ![]() cytoplasm organization / cytolytic granule membrane / plus-end-directed vesicle transport along microtubule / mitocytosis / anterograde dendritic transport of neurotransmitter receptor complex / anterograde neuronal dense core vesicle transport / anterograde axonal protein transport / retrograde neuronal dense core vesicle transport / vesicle transport along microtubule / lysosome localization ...cytoplasm organization / cytolytic granule membrane / plus-end-directed vesicle transport along microtubule / mitocytosis / anterograde dendritic transport of neurotransmitter receptor complex / anterograde neuronal dense core vesicle transport / anterograde axonal protein transport / retrograde neuronal dense core vesicle transport / vesicle transport along microtubule / lysosome localization / positive regulation of potassium ion transport / Kinesins / plus-end-directed microtubule motor activity / RHO GTPases activate KTN1 / stress granule disassembly / positive regulation of axon guidance / mitochondrion transport along microtubule / COPI-dependent Golgi-to-ER retrograde traffic / centrosome localization / microtubule motor activity / ciliary rootlet / natural killer cell mediated cytotoxicity / kinesin complex / synaptic vesicle transport / Insulin processing / microtubule-based movement / microtubule-based process / centriolar satellite / axon cytoplasm / phagocytic vesicle / MHC class II antigen presentation / dendrite cytoplasm / regulation of membrane potential / axon guidance / positive regulation of synaptic transmission, GABAergic / positive regulation of protein localization to plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / cellular response to type II interferon / microtubule cytoskeleton organization / microtubule cytoskeleton / Signaling by ALK fusions and activated point mutants / mitotic cell cycle / nervous system development / microtubule binding / vesicle / microtubule / hydrolase activity / cadherin binding / protein heterodimerization activity / GTPase activity / protein-containing complex binding / GTP binding / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / membrane / metal ion binding / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 9.0 Å | |||||||||
![]() | Sindelar CV / Downing KH | |||||||||
![]() | ![]() Title: The beginning of kinesin's force-generating cycle visualized at 9-A resolution. Authors: Charles V Sindelar / Kenneth H Downing / ![]() Abstract: We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, ...We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in "ejecting" adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the "neck linker"). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself. | |||||||||
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 | ![]() | 15.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.5 KB 12.5 KB | Display Display | ![]() |
Images | ![]() | 90.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2p4nMC M: atomic model generated by this map 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 | 9-Angstrom map of nucleotide-free kinesin complexed to the microtubule | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 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 : Nucleotide-free kinesin bound to a 13-protofilament microtubule
Entire | Name: Nucleotide-free kinesin bound to a 13-protofilament microtubule |
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Components |
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-Supramolecule #1000: Nucleotide-free kinesin bound to a 13-protofilament microtubule
Supramolecule | Name: Nucleotide-free kinesin bound to a 13-protofilament microtubule type: sample / ID: 1000 Oligomeric state: The 13-protofilament microtubule forms a pseudo helix interrupted by a single seam Number unique components: 3 |
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-Macromolecule #1: Kinesin
Macromolecule | Name: Kinesin / type: protein_or_peptide / ID: 1 / Name.synonym: K349 Details: C220 "cys-lite" K349 construct: 6 of 8 native cysteines eliminated, introduced cysteine at position 220 Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 36.4134 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #2: Alpha-tubulin
Macromolecule | Name: Alpha-tubulin / type: protein_or_peptide / ID: 2 / Details: Glycerol-free tubulin purchased from Cytoskeleton / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 50.1279 KDa |
-Macromolecule #3: Beta-tubulin
Macromolecule | Name: Beta-tubulin / type: protein_or_peptide / ID: 3 / Details: Glycerol-free tubulin purchased from Cytoskeleton / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 49.9283 KDa |
-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: 25mM Pipes, 25mM NaCl, 2mM MgCl2, 1mM EGTA |
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Grid | Details: 300 mesh copper grid |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Home-built Method: Grids were not glow discharged. Liquid was almost entirely wicked away prior to blotting. Blotting time was less than 0.5 sec. |
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Electron microscopy
Microscope | JEOL 4000EX |
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Temperature | Average: 105 K |
Alignment procedure | Legacy - Astigmatism: e.g objective lens astigmatism was corrected at 400,000 times magnification |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6.3 µm / Number real images: 350 / Average electron dose: 16 e/Å2 / Bits/pixel: 16 |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 400 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.1 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 60000 |
Sample stage | Specimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN |
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Image processing
CTF correction | Details: CTF correction was integrated into the back-projection process |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER Details: Data from 188 microtubules were incorporated into the final reconstruction Number images used: 150000 |
-Atomic model buiding 1
Initial model | (PDB ID: , ) |
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Software | Name: SITUS |
Details | Protocol: Rigid body. Kinesin and tubulin were separately fitted into the final map using the program SITUS. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation |
Output model | ![]() PDB-2p4n: |