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Yorodumi- EMDB-6354: Asymmetric (C1) reconstruction of EB3-bound microtubule (merged d... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6354 | |||||||||
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Title | Asymmetric (C1) reconstruction of EB3-bound microtubule (merged dataset containing tubulin bound to GTPgammaS, GMPCPP and GDP) | |||||||||
Map data | Asymmetric (C1) reconstruction of EB3-bound microtubule (merged dataset containing tubulin bound to GTPgammaS, GMPCPP and GDP) | |||||||||
Sample |
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Keywords | microtubule / EB3 | |||||||||
Function / homology | Function and homology information 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 / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / 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 / spindle midzone / spindle assembly / regulation of microtubule polymerization or depolymerization / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / protein localization / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / 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 | Sus scrofa (pig) / Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.7 Å | |||||||||
Authors | Zhang R / Alushin GM / Brown A / Nogales E | |||||||||
Citation | Journal: Cell / Year: 2015 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6354.map.gz | 96.2 MB | EMDB map data format | |
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Header (meta data) | emd-6354-v30.xml emd-6354.xml | 13.1 KB 13.1 KB | Display Display | EMDB header |
Images | 400_6354.gif 80_6354.gif | 161.4 KB 8.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6354 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6354 | HTTPS FTP |
-Validation report
Summary document | emd_6354_validation.pdf.gz | 372 KB | Display | EMDB validaton report |
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Full document | emd_6354_full_validation.pdf.gz | 371.6 KB | Display | |
Data in XML | emd_6354_validation.xml.gz | 8.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6354 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6354 | HTTPS FTP |
-Related structure data
Related structure data | 3jawMC 6347C 6348C 6349C 6350C 6351C 6352C 6353C 6355C 3jakC 3jalC 3jarC 3jasC 3jatC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_6354.map.gz / Format: CCP4 / Size: 500 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Asymmetric (C1) reconstruction of EB3-bound microtubule (merged dataset containing tubulin bound to GTPgammaS, GMPCPP and GDP) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.32 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : EB3-bound microtubule (merged dataset containing tubulin bound to...
Entire | Name: EB3-bound microtubule (merged dataset containing tubulin bound to GTPgammaS, GMPCPP, and GDP) |
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Components |
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-Supramolecule #1000: EB3-bound microtubule (merged dataset containing tubulin bound to...
Supramolecule | Name: EB3-bound microtubule (merged dataset containing tubulin bound to GTPgammaS, GMPCPP, and GDP) type: sample / ID: 1000 Details: Merged dataset containing tubulin bound to GTPgammaS, GMPCPP, and GDP Oligomeric state: helical assembly / Number unique components: 3 |
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-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 |
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Source (natural) | Organism: Sus scrofa (pig) / synonym: Porcine / Tissue: Brain / Organelle: Cytoplasm / Location in cell: Cytoskeleton |
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 |
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Source (natural) | Organism: Sus scrofa (pig) / synonym: Porcine / Tissue: Brain / Organelle: Cytoplasm / Location in cell: Cytoskeleton |
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 |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Organelle: Cytoplasm / Location in cell: Cytoskeleton |
Molecular weight | Theoretical: 23 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21-CodonPlus-(DE3)-RIL / Recombinant plasmid: 2BT |
Sequence | UniProtKB: Microtubule-associated protein RP/EB family member 3 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | helical array |
-Sample preparation
Concentration | 1.5 mg/mL |
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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 |
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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: 922 / 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: FIELD EMISSION GUN |
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
CTF correction | Details: CTFFIND4, each particle |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 9.44 Å Applied symmetry - Helical parameters - Δ&Phi: 27.71 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: OTHER / Software - Name: EMAN1, FREALIGN / Details: asymmetric (C1) reconstruction / Number images used: 79702 |