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- EMDB-7101: TPX2_mini decorated GMPCPP-microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-7101
TitleTPX2_mini decorated GMPCPP-microtubule
Map dataTPX2_mini decorated GMPCPP- microtubule
Sample
  • Complex: TPX2_mini decorated GMPCPP-microtubule
    • Complex: Tubulin
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta chain
    • Complex: Targeting protein for Xklp2
      • Protein or peptide: Targeting protein for Xklp2
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
Keywordsmitosis / GanGTP / nucleation / CELL CYCLE
Function / homology
Function and homology information


axon hillock / negative regulation of microtubule depolymerization / microtubule nucleation / importin-alpha family protein 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 ...axon hillock / negative regulation of microtubule depolymerization / microtubule nucleation / importin-alpha family protein 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 / COPI-mediated anterograde transport / activation of protein kinase activity / intercellular bridge / mitotic spindle assembly / regulation of mitotic spindle organization / AURKA Activation by TPX2 / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / spindle pole / spindle / microtubule cytoskeleton / mitotic cell cycle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Regulation of TP53 Activity through Phosphorylation / microtubule / molecular adaptor activity / cell division / GTPase activity / GTP binding / apoptotic process / protein kinase binding / nucleoplasm / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
TPX2 / Aurora-A binding / TPX2, C-terminal / TPX2 central domain / Targeting protein for Xklp2 (TPX2) domain / Aurora-A binding / Cell cycle regulated microtubule associated protein / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site ...TPX2 / Aurora-A binding / TPX2, C-terminal / TPX2 central domain / Targeting protein for Xklp2 (TPX2) domain / Aurora-A binding / Cell cycle regulated microtubule associated protein / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Tubulin beta chain / Tubulin alpha-1B chain / Targeting protein for Xklp2
Similarity search - Component
Biological speciessus scrofa (pig) / Homo sapiens (human) / Sus scrofa (pig)
Methodhelical reconstruction / cryo EM / Resolution: 3.3 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
Howard Hughes Medical Institute (HHMI) United States
Cancer Research UKFC001163 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001163 United Kingdom
Wellcome TrustFC001163 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-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing 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
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  • Surface view colored by cylindrical radius
  • Surface level: 3.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6bjc
  • Surface level: 3.7
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6bjc
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7101.png

Downloads & links

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Map

FileDownload / File: emd_7101.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTPX2_mini 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-12.16531 - 25.345735999999999
Average (Standard dev.)-0.016123138 (±0.93212706)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-12.16525.346-0.016

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Supplemental data

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Sample components

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Entire : TPX2_mini decorated GMPCPP-microtubule

EntireName: TPX2_mini decorated GMPCPP-microtubule
Components
  • Complex: TPX2_mini decorated GMPCPP-microtubule
    • Complex: Tubulin
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta chain
    • Complex: Targeting protein for Xklp2
      • Protein or peptide: Targeting protein for Xklp2
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

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Supramolecule #1: TPX2_mini decorated GMPCPP-microtubule

SupramoleculeName: TPX2_mini decorated GMPCPP-microtubule / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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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) / Organ: brain

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Supramolecule #3: Targeting protein for Xklp2

SupramoleculeName: Targeting protein for Xklp2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
Molecular weightTheoretical: 50.204445 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
Molecular weightTheoretical: 49.90777 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEGEEDEA

UniProtKB: Tubulin beta chain

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Macromolecule #3: Targeting protein for Xklp2

MacromoleculeName: Targeting protein for Xklp2 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 85.811328 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
SequenceString: MSQVKSSYSY DAPSDFINFS SLDDEGDTQN IDSWFEEKAN LENKLLGKNG TGGLFQGKTP LRKANLQQAI VTPLKPVDNT YYKEAEKEN LVEQSIPSNA CSSLEVEAAI SRKTPAQPQR RSLRLSAQKD LEQKEKHHVK MKAKRCATPV IIDEILPSKK M KVSNNKKK ...String:
MSQVKSSYSY DAPSDFINFS SLDDEGDTQN IDSWFEEKAN LENKLLGKNG TGGLFQGKTP LRKANLQQAI VTPLKPVDNT YYKEAEKEN LVEQSIPSNA CSSLEVEAAI SRKTPAQPQR RSLRLSAQKD LEQKEKHHVK MKAKRCATPV IIDEILPSKK M KVSNNKKK PEEEGSAHQD TAEKNASSPE KAKGRHTVPC MPPAKQKFLK STEEQELEKS MKMQQEVVEM RKKNEEFKKL AL AGIGQPV KKSVSQVTKS VDFHFRTDER IKQHPKNQEE YKEVNFTSEL RKHPSSPARV TKGCTIVKPF NLSQGKKRTF DET VSTYVP LAQQVEDFHK RTPNRYHLRS KKDDINLLPS KSSVTKICRD PQTPVLQTKH RARAVTCKST AELEAEELEK LQQY KFKAR ELDPRILEGG PILPKKPPVK PPTEPIGFDL EIEKRIQERE SKKKTEDEHF EFHSRPCPTK ILEDVVGVPE KKVLP ITVP KSPAFALKNR IRMPTKEDEE EDEPVVIKAQ PVPHYGVPFK PQIPEARTVE ICPFSFDSRD KERQLQKEKK IKELQK GEV PKFKALPLPH FDTINLPEKK VKNVTQIEPF CLETDRRGAL KAQTWKHQLE EELRQQKEAA CFKARPNTVI SQEPFVP KK EKKSVAEGLS GSLVQEPFQL ATEKRAKERQ ELEKRMAEVE AQKAQQLEEA RLQEEEQKKE ELARLRRELV HKANPIRK Y QGLEIKSSDQ PLTVPVSPKF STRFHC

UniProtKB: Targeting protein for Xklp2

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 6 / Number of copies: 6 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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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 KRIOS
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: 0.01 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 9.03 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: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 85000
Startup modelType of model: EMDB MAP
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-6bjc:
TPX2_mini decorated GMPCPP-microtubule

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