+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-7102 | ||||||||||||||||||
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Title | TPX2_micro decorated GMPCPP-microtubule | ||||||||||||||||||
Map data | TPX2_micro decorated GMPCPP-microtubule | ||||||||||||||||||
Sample |
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Biological species | sus scrofa (pig) / Homo sapiens (human) / Sus scrofa (pig) | ||||||||||||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.6 Å | ||||||||||||||||||
Authors | Zhang R / Nogales E | ||||||||||||||||||
Funding support | United States, United Kingdom, 5 items
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Citation | Journal: Elife / Year: 2017 Title: Structural insight into TPX2-stimulated microtubule assembly. Authors: Rui Zhang / Johanna Roostalu / Thomas Surrey / Eva Nogales / Abstract: During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The ...During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation. | ||||||||||||||||||
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_7102.map.gz | 156 MB | EMDB map data format | |
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Header (meta data) | emd-7102-v30.xml emd-7102.xml | 15.5 KB 15.5 KB | Display Display | EMDB header |
Images | emd_7102.png | 395.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-7102 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-7102 | HTTPS FTP |
-Related 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_7102.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | TPX2_micro decorated GMPCPP-microtubule | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.33 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 : TPX2_micro decorated GMPCPP-microtubule
Entire | Name: TPX2_micro decorated GMPCPP-microtubule |
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Components |
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-Supramolecule #1: TPX2_micro decorated GMPCPP-microtubule
Supramolecule | Name: TPX2_micro decorated GMPCPP-microtubule / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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-Supramolecule #2: tubulin
Supramolecule | Name: tubulin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: sus scrofa (pig) |
-Supramolecule #3: TPX2
Supramolecule | Name: TPX2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths) Recombinant strain: sf21 |
-Macromolecule #1: alpha tubulin
Macromolecule | Name: alpha tubulin / type: other / ID: 1 / Classification: other |
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Source (natural) | Organism: Sus scrofa (pig) / Organ: brain |
Sequence | String: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT SLLMERLSVD YGKKSKLEFS ...String: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT SLLMERLSVD YGKKSKLEFS IYPAPQVSTA VVEPYNSILT THTTLEHSDC AFMVDNEAIY DICRRNLDIE RPTYTNLNRL ISQIVSSITA SLRFDGALNV DLTEFQTNLV PYPRIHFPLA TYAPVISAEK AYHEQLSVAE ITNACFEPAN QMVKCDPRHG KYMACCLLYR GDVVPKDVNA AIATIKTKRS IQFVDWCPTG FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHKFDLMYA KRAFVHWYVG EGMEEGEFSE AREDMAALEK DYEEVGVDSV EGEGEEEGEE Y |
-Macromolecule #2: beta tubulin
Macromolecule | Name: beta tubulin / type: other / ID: 2 / Classification: other |
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Source (natural) | Organism: Sus scrofa (pig) / Organ: brain |
Sequence | String: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL LISKIREEYP DRIMNTFSVV ...String: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL LISKIREEYP DRIMNTFSVV PSPKVSDTVV EPYNATLSVH QLVENTDETY CIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTCL RFPGQLNADL RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQMFDAKNMM AACDPRHGRY LTVAAVFRGR MSMKEVDEQM LNVQNKNSSY FVEWIPNNVK TAVCDIPPRG LKMSATFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG EGMDEMEFTE AESNMNDLVS EYQQYQDATA DEQGEFEEEG EEDEA |
-Macromolecule #3: TPX2
Macromolecule | Name: TPX2 / type: other / ID: 3 / Classification: other |
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Source (natural) | Organism: Homo sapiens (human) |
Sequence | String: MSQVKSSYSY DAPSDFINFS SLDDEGDTQN IDSWFEEKAN LENKLLGKNG TGGLFQGKTP LRKANLQQA IVTPLKPVDN TYYKEAEKEN LVEQSIPSNA CSSLEVEAAI SRKTPAQPQR R SLRLSAQK DLEQKEKHHV KMKAKRCATP VIIDEILPSK KMKVSNNKKK ...String: MSQVKSSYSY DAPSDFINFS SLDDEGDTQN IDSWFEEKAN LENKLLGKNG TGGLFQGKTP LRKANLQQA IVTPLKPVDN TYYKEAEKEN LVEQSIPSNA CSSLEVEAAI SRKTPAQPQR R SLRLSAQK DLEQKEKHHV KMKAKRCATP VIIDEILPSK KMKVSNNKKK PEEEGSAHQD TA EKNASSP EKAKGRHTVP CMPPAKQKFL KSTEEQELEK SMKMQQEVVE MRKKNEEFKK LAL AGIGQP VKKSVSQVTK SVDFHFRTDE RIKQHPKNQE EYKEVNFTSE LRKHPSSPAR VTKG CTIVK PFNLSQGKKR TFDETVSTYV PLAQQVEDFH KRTPNRYHLR SKKDDINLLP SKSSV TKIC RDPQTPVLQT KHRARAVTCK STAELEAEEL EKLQQYKFKA RELDPRILEG GPILPK KPP VKPPTEPIGF DLEIEKRIQE RESKKKTEDE HFEFHSRPCP TKILEDVVGV PEKKVLP IT VPKSPAFALK NRIRMPTKED EEEDEPVVIK AQPVPHYGVP FKPQIPEART VEICPFSF D SRDKERQLQK EKKIKELQKG EVPKFKALPL PHFDTINLPE KKVKNVTQIE PFCLETDRR GALKAQTWKH QLEEELRQQK EAACFKARPN TVISQEPFVP KKEKKSVAEG LSGSLVQEPF QLATEKRAK ERQELEKRMA EVEAQKAQQL EEARLQEEEQ KKEELARLRR ELVHKANPIR K YQGLEIKS SDQPLTVPVS PKFSTRFHC |
Recombinant expression | Organism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | helical array |
-Sample preparation
Buffer | pH: 6.8 / Details: BRB80 |
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Grid | Model: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK II / Details: blot for 4 seconds before plunging. |
-Electron microscopy
Microscope | FEI TITAN |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm |
Sample stage | Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 4 / Number real images: 2000 / Average exposure time: 6.0 sec. / Average electron dose: 27.6 e/Å2 |
-Image processing
CTF correction | Software - Name: CTFFIND |
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Final angle assignment | Type: NOT APPLICABLE / Software - Name: FREALIGN |
Final reconstruction | Applied symmetry - Helical parameters - Δz: 9.04 Å Applied symmetry - Helical parameters - Δ&Phi: -25.74 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 85000 |
-Atomic model buiding 1
Refinement | Protocol: AB INITIO MODEL |
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