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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-5895 | |||||||||
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Title | Cryo EM density of microtubules stabilized by GMPCPP | |||||||||
![]() | cryo-EM reconstruction of microtubule stabilized by GMPCPP | |||||||||
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Function / homology | ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Alushin GM / Lander GC / Kellogg EH / Zhang R / Baker D / Nogales E | |||||||||
![]() | ![]() Title: High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Authors: Gregory M Alushin / Gabriel C Lander / Elizabeth H Kellogg / Rui Zhang / David Baker / Eva Nogales / ![]() Abstract: Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is ...Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces. | |||||||||
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 | ![]() | 4.1 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.5 KB 13.5 KB | Display Display | ![]() |
Images | ![]() ![]() | 58.4 KB 4.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3j6eMC ![]() 5896C ![]() 5897C ![]() 5898C ![]() 5899C ![]() 3j6fC ![]() 3j6gC 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 | cryo-EM reconstruction of microtubule stabilized by GMPCPP | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.74 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 : microtubule stabilized by GMPCPP
Entire | Name: microtubule stabilized by GMPCPP |
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Components |
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-Supramolecule #1000: microtubule stabilized by GMPCPP
Supramolecule | Name: microtubule stabilized by GMPCPP / type: sample / ID: 1000 / Number unique components: 3 |
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-Macromolecule #1: Alpha tubulin
Macromolecule | Name: Alpha tubulin / type: protein_or_peptide / ID: 1 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 55 KDa |
-Macromolecule #2: Beta tubulin
Macromolecule | Name: Beta tubulin / type: protein_or_peptide / ID: 2 / Details: Beta subunit is bound to GMPCPP / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 55 KDa |
-Macromolecule #3: kinesin
Macromolecule | Name: kinesin / type: protein_or_peptide / ID: 3 Details: Human monomeric kinesin K349 cys-lite described in: Rice, S., Lin, A.W., Safer, D., Hart, C.L., Naber, N., Carragher, B.O., Cain, S.M., Pechatnikova, E., Wilson-Kubalek, E.M., Whittaker, M., ...Details: Human monomeric kinesin K349 cys-lite described in: Rice, S., Lin, A.W., Safer, D., Hart, C.L., Naber, N., Carragher, B.O., Cain, S.M., Pechatnikova, E., Wilson-Kubalek, E.M., Whittaker, M., et al. (1999). A structural change in the kinesin motor protein that drives motility. Nature 402, 778-784. Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 36 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | helical array |
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Sample preparation
Concentration | 0.25 mg/mL |
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Buffer | pH: 6.8 Details: 80mM PIPES, 1mM EGTA, 1mM MgCl2, 1mM DTT, 0.05% Nonidet P-40 |
Grid | Details: 400 mesh C-flat 1.2/1.3, glow discharged in Edwards carbon evaporator |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90.4 K / Instrument: FEI VITROBOT MARK II Method: 4 uL microtubules were applied to grid for 30-60 seconds, 4 uL kinesin was added and incubated for 30 seconds, 4 uL buffer was removed, then another 4 uL kinesin was added and incubated for ...Method: 4 uL microtubules were applied to grid for 30-60 seconds, 4 uL kinesin was added and incubated for 30 seconds, 4 uL buffer was removed, then another 4 uL kinesin was added and incubated for 30 seconds. 4 uL buffer was removed, then the grid was blotted for 2 seconds before plunging. |
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Electron microscopy
Microscope | FEI TITAN |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 72000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder: Gatan 626 holder / Specimen holder model: GATAN LIQUID NITROGEN |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnifacation. |
Date | Apr 26, 2012 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6.35 µm / Number real images: 252 / Average electron dose: 25.0 e/Å2 / Bits/pixel: 16 |
Tilt angle min | 0 |
Tilt angle max | 0 |
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Image processing
CTF correction | Details: ctftilt |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 8.9 Å Applied symmetry - Helical parameters - Δ&Phi: 25.75 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: OTHER / Software - Name: FREALIGN / Number images used: 57451 |
Details | Initial alignments performed with EMAN2/SPARX, including refinement of helical parameters with the IHRSR programs of Egelman. Final alignment and reconstruction performed with FREALIGN. |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: ROSETTA |
Details | Structure represents the minimized average structure of the 1% lowest energy structures from the refinement run. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | ![]() PDB-3j6e: |