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- EMDB-7102: TPX2_micro decorated GMPCPP-microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-7102
TitleTPX2_micro decorated GMPCPP-microtubule
Map dataTPX2_micro decorated GMPCPP-microtubule
Sample
  • Complex: TPX2_micro decorated GMPCPP-microtubule
    • Complex: tubulin
      • Other: alpha tubulin
      • Other: beta tubulin
    • Complex: TPX2
      • Other: TPX2
Biological speciessus scrofa (pig) / Homo sapiens (human) / Sus scrofa (pig)
Methodhelical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZhang R / Nogales E
Funding support United States, United Kingdom, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM051487 United States
Cancer Research UKFC001163 United Kingdom
Howard Hughes Medical Institute (HHMI) United States
Wellcome TrustFC001163 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001163 United Kingdom
CitationJournal: 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
DepositionNov 5, 2017-
Header (metadata) releaseNov 15, 2017-
Map releaseNov 22, 2017-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7102.png

Downloads & links

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Map

FileDownload / File: emd_7102.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTPX2_micro decorated GMPCPP-microtubule
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 512 pix.
= 680.96 Å		1.33 Å/pix.
x 512 pix.
= 680.96 Å		1.33 Å/pix.
x 512 pix.
= 680.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.33 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.7 / Movie #1: 3.7
Minimum - Maximum-9.645132 - 20.603985
Average (Standard dev.)-0.021339504 (±0.9093206)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 680.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.331.331.33
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z680.960680.960680.960
α/β/γ90.00090.00090.000
start NX/NY/NZ-51-35-11
NX/NY/NZ11110799
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-9.64520.604-0.021

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Supplemental data

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Sample components

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Entire : TPX2_micro decorated GMPCPP-microtubule

EntireName: TPX2_micro decorated GMPCPP-microtubule
Components
  • Complex: TPX2_micro decorated GMPCPP-microtubule
    • Complex: tubulin
      • Other: alpha tubulin
      • Other: beta tubulin
    • Complex: TPX2
      • Other: TPX2

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Supramolecule #1: TPX2_micro decorated GMPCPP-microtubule

SupramoleculeName: TPX2_micro decorated GMPCPP-microtubule / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: tubulin

SupramoleculeName: tubulin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: sus scrofa (pig)

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Supramolecule #3: TPX2

SupramoleculeName: TPX2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
Recombinant strain: sf21

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Macromolecule #1: alpha tubulin

MacromoleculeName: alpha tubulin / type: other / ID: 1 / Classification: other
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
SequenceString: 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

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Macromolecule #2: beta tubulin

MacromoleculeName: beta tubulin / type: other / ID: 2 / Classification: other
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
SequenceString: 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

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Macromolecule #3: TPX2

MacromoleculeName: TPX2 / type: other / ID: 3 / Classification: other
Source (natural)Organism: Homo sapiens (human)
SequenceString: 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 expressionOrganism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 6.8 / Details: BRB80
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK II / Details: blot for 4 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionApplied 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
CTF correctionSoftware - Name: CTFFIND
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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