[English] 日本語
![](img/lk-miru.gif)
- EMDB-6349: Cryo-EM structure of GTPgammaS-microtubule co-polymerized with EB... -
+
Open data
-
Basic information
Entry | Database: EMDB / ID: EMD-6349 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of GTPgammaS-microtubule co-polymerized with EB3 (merged dataset with and without kinesin bound) | |||||||||
![]() | Reconstruction of GTPgammaS-microtubule co-polymerized with EB3 (merged dataset with and without kinesin bound) | |||||||||
![]() |
| |||||||||
![]() | microtubule / EB3 / GTPgammaS / kinesin | |||||||||
Function / homology | ![]() mitotic spindle astral microtubule end / protein localization to microtubule / microtubule plus-end / microtubule plus-end binding / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin ...mitotic spindle astral microtubule end / protein localization to microtubule / microtubule plus-end / microtubule plus-end binding / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / microtubule organizing center / COPI-mediated anterograde transport / regulation of microtubule polymerization / positive regulation of cyclin-dependent protein serine/threonine kinase activity / positive regulation of protein kinase activity / regulation of microtubule polymerization or depolymerization / spindle assembly / spindle midzone / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / protein localization / mitotic cell cycle / midbody / microtubule binding / microtubule / cell division / GTPase activity / GTP binding / protein kinase binding / perinuclear region of cytoplasm / positive regulation of DNA-templated transcription / identical protein binding / metal ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
![]() | Zhang R / Alushin GM / Brown A / Nogales E | |||||||||
![]() | ![]() Title: Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins. Authors: Rui Zhang / Gregory M Alushin / Alan Brown / Eva Nogales / ![]() ![]() Abstract: Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo- ...Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an "anchor point," leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics. | |||||||||
History |
|
-
Structure visualization
Movie |
![]() |
---|---|
Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
-
Downloads & links
-EMDB archive
Map data | ![]() | 156.6 MB | ![]() | |
---|---|---|---|---|
Header (meta data) | ![]() ![]() | 14.4 KB 14.4 KB | Display Display | ![]() |
Images | ![]() ![]() | 161.8 KB 7.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3jakMC ![]() 6347C ![]() 6348C ![]() 6350C ![]() 6351C ![]() 6352C ![]() 6353C ![]() 6354C ![]() 6355C ![]() 3jalC ![]() 3jarC ![]() 3jasC ![]() 3jatC ![]() 3jawC M: atomic model generated by this map C: citing same article ( |
---|---|
Similar structure data |
-
Links
EMDB pages | ![]() ![]() |
---|---|
Related items in Molecule of the Month |
-
Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Reconstruction of GTPgammaS-microtubule co-polymerized with EB3 (merged dataset with and without kinesin bound) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.32 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-
Sample components
-Entire : EB3- and kinesin-bound GTPgammaS microtubule
Entire | Name: EB3- and kinesin-bound GTPgammaS microtubule |
---|---|
Components |
|
-Supramolecule #1000: EB3- and kinesin-bound GTPgammaS microtubule
Supramolecule | Name: EB3- and kinesin-bound GTPgammaS microtubule / type: sample / ID: 1000 Details: GTPgammaS microtubules were polymerized on the EM grid in the presence of GMPCPP-MT seeds and excess EB3, then washed with kinesin. Oligomeric state: helical assembly / Number unique components: 4 |
---|
-Macromolecule #1: Alpha tubulin
Macromolecule | Name: Alpha tubulin / type: protein_or_peptide / ID: 1 Details: Porcine tubulin powder was purchased from Cytoskeleton Inc. Oligomeric state: Helical assembly / Recombinant expression: No / Database: NCBI |
---|---|
Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 55 KDa |
Sequence | UniProtKB: Tubulin alpha-1B chain |
-Macromolecule #2: Beta tubulin
Macromolecule | Name: Beta tubulin / type: protein_or_peptide / ID: 2 Details: Porcine tubulin powder was purchased from Cytoskeleton Inc. Oligomeric state: Helical assembly / Recombinant expression: No / Database: NCBI |
---|---|
Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 55 KDa |
Sequence | UniProtKB: Tubulin beta chain |
-Macromolecule #3: EB3
Macromolecule | Name: EB3 / type: protein_or_peptide / ID: 3 / Name.synonym: MAPRE3 Details: A monomeric construct containing residues 1-200, which lacks the C-terminal dimerization domain Oligomeric state: Dimer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 23 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Microtubule-associated protein RP/EB family member 3 |
-Macromolecule #4: kinesin
Macromolecule | Name: kinesin / type: protein_or_peptide / ID: 4 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 |
---|---|
Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 36 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
![]() | helical reconstruction |
Aggregation state | helical array |
-
Sample preparation
Concentration | 1.5 mg/mL |
---|---|
Buffer | pH: 6.8 Details: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT, 0.05% Nonidet P-40 |
Grid | Details: 400 mesh C-flat 1.2/1.3 EM grid, glow discharged in Ar/O2 gas (Solarus, Gatan Inc) |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 90.4 K / Instrument: FEI VITROBOT MARK II / Method: Blot once for 4 seconds before plunging. |
-
Electron microscopy
Microscope | FEI TITAN |
---|---|
Temperature | Average: 90 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 27,500 times magnification. |
Details | The camera was operated in counting mode, with a dose rate of ~8 electrons/pixel/s on the camera. A total exposure time of 6 s was fractionated into 20 movie frames. |
Date | May 16, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 345 / Average electron dose: 27.6 e/Å2 Details: The camera was operated in counting mode, with a dose rate of ~8 electrons/pixel/s on the camera. A total exposure time of 6 s was fractionated into 20 movie frames. |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 27500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 27500 |
Sample stage | Specimen holder: Gatan 626 holder / Specimen holder model: GATAN LIQUID NITROGEN |
-
Image processing
Details | IHRSR algorithm with microtubule-specific pseudo-helical symmetry applied |
---|---|
Final reconstruction | Applied symmetry - Helical parameters - Δz: 9.45 Å Applied symmetry - Helical parameters - Δ&Phi: 27.72 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: OTHER / Software - Name: EMAN1, FREALIGN Details: Pseudo-helical symmetry was applied during the reconstruction step. |
CTF correction | Details: CTFFIND4, each particle |