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- PDB-7sj8: 13pf wildtype microtubule from recombinant human tubulin decorate... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7sj8 | ||||||
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Title | 13pf wildtype microtubule from recombinant human tubulin decorated with kinesin | ||||||
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Function / homology | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ![]() ![]() | ||||||
![]() | LaFrance, B.J. / Greber, B.J. / Zhang, R. / McCollum, C. / Nogales, E. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules. Authors: Benjamin J LaFrance / Johanna Roostalu / Gil Henkin / Basil J Greber / Rui Zhang / Davide Normanno / Chloe O McCollum / Thomas Surrey / Eva Nogales / ![]() ![]() ![]() Abstract: Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is ...Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability. #1: ![]() Title: Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules Authors: LaFrance, B.J. / Roostalu, J. / Henkin, G. / Greber, B.J. / Zhang, R. / Normanno, D. / McCollum, C. / Surrey, T. / Nogales, E. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 875.6 KB | Display | ![]() |
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PDB format | ![]() | 735.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 25157MC ![]() 7sj7C ![]() 7sj9C ![]() 7sjaC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 51033.324 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #2: Protein | Mass: 51276.367 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #3: Chemical | ChemComp-GTP / ![]() #4: Chemical | ChemComp-MG / #5: Chemical | ChemComp-GDP / ![]() Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction |
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Sample preparation
Component | Name: 13pf wildtype microtubule from recombinant human tubulin and decorated with kinesin Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 0.102 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() ![]() |
Buffer solution | pH: 6.8 Details: 80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 2 mM GTP, pH 6.8 |
Specimen | Conc.: 4.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() Details: 44 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3 |
Vitrification![]() | Cryogen name: ETHANE-PROPANE Details: 30 second adsorption time onto grid. blot force 15, 4 second blot time, 37 degrees, and 100 percent humidity. 0.5 second drain time. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() ![]() |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.18_3845: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Images collected in super-resolution mode, Motioncorr2 5x5 binning of frames | ||||||||||||||||||||||||||||||||||||||||
CTF correction![]() | Details: CTFFind4 and CTFRefine were used within the RELION3.1 framework. Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: -0.09 ° / Axial rise/subunit: 81.69 Å / Axial symmetry: C1 | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Details: Microtubules were picked within RELION 3.1 helical picking functionality and extracted along the filament with an 82A sliding box. | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23364 / Num. of class averages: 1 / Symmetry type: HELICAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 115 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation Coefficient | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6DPU Pdb chain-ID: A / Accession code: 6DPU / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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