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- PDB-8cx6: TPX2 Minimal Active Domain on Microtubules -

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ID or keywords:

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Basic information

Entry
Database: PDB / ID: 8cx6
TitleTPX2 Minimal Active Domain on Microtubules
ComponentsTargeting protein for Xklp2-A
KeywordsCELL CYCLE / spindle assembly factor / branching microtubule nucleation
Function / homology
Function and homology information


regulation of mitotic spindle organization / spindle pole / microtubule / cell cycle / cell division / nucleus / 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
Similarity search - Domain/homology
Targeting protein for Xklp2-A
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
MethodSOLID-STATE NMR / simulated annealing
AuthorsGuo, C. / Alfaro-Aco, R. / Russell, R. / Zhang, C. / Petry, S. / Polenova, T.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM123493 United States
The Pew Charitable Trusts00027340 United States
David and Lucile Packard Foundation201440376 United States
National Science Foundation (NSF, United States)CHE-0959496 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30GM110758 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of protein condensation on microtubules underlying branching microtubule nucleation.
Authors: Guo, C. / Alfaro-Aco, R. / Zhang, C. / Russell, R.W. / Petry, S. / Polenova, T.
History
DepositionMay 19, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Targeting protein for Xklp2-A


Theoretical massNumber of molelcules
Total (without water)28,4441
Polymers28,4441
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 100structures with the lowest energy
RepresentativeModel #1medoid

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Components

#1: Protein Targeting protein for Xklp2-A


Mass: 28443.770 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: tpx2-a / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2 / References: UniProt: Q6NUF4

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Experimental details

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Experiment

ExperimentMethod: SOLID-STATE NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
212isotropic22D NH HETCOR
222isotropic22D CH HETCOR
232isotropic23D (H)CANH
242isotropic23D (H)(CO)CA(CO)NH
252isotropic23D (H)CONH
2122isotropic22D RFDR 2.4 ms
3182isotropic22D CH HETCOR (2.1 ms/2.1 ms CP)
3172isotropic22D CH HETCOR (2.8 ms/2.7 ms CP)
2162isotropic22D CH HETCOR
4152isotropic22D CH HETCOR
1141isotropic12D CORD (50ms mixing)
1131isotropic12D CORD (200ms mixing)
1231isotropic12D RFDR
1221isotropic12D NCA
1211isotropic12D HN HETCOR
5201isotropic12D CH HETCOR
5191isotropic12D CH HETCOR
5291isotropic12D NH HETCOR
5281isotropic13D (H)CANH
5271isotropic13D (H)CONH
5261isotropic12D RFDR
5251isotropic12D CORD (200ms mixing)
5241isotropic12D NCACX
5301isotropic12D NCOCX

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solid11.5 mM [U-13C; U-15N] TPX2a5-7, 100 uM Microtubules, 25 mM phosphate buffer, 100% H2OThe TPX2 protein is fully protonated uniformly labeled with U-[13C6]-glucose and 15NH4Cl. The paclitaxel-stabilized microtubules are polymerized from bovine brain tubulin in BRB80 buffer. The mixture of TPX2 and microtubules were centrifuged through a 40% glycerol-BRB80 cushion.protonated U-[13C,15N]-TPX2a5-7/MT assemblies100% H2O
solid21.5 mM [U-13C; U-15N],D; 100% back-exchanged TPX2a5-7, 100 uM Microtubules, 25 mM phosphate buffer, 100% H2OThe deuterated U-[13C,15N]-labeled TPX2a5-7 was overexpressed in minimal media in 100% D2O supplemented with 15NH4Cl and U-[13C6, D7]-glucose. The exchangeable sites in TPX2 protein was back-exchanged in 100% H2O. The paclitaxel-stabilized microtubules are polymerized from bovine brain tubulin in BRB80 buffer. The mixture of TPX2 and microtubules were centrifuged through a 40% glycerol-BRB80 cushion.deuterated U-[13C,15N]-labeled TPX2a5-7/MT assemblies100% H2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.5 mMTPX2a5-7[U-13C; U-15N]1
100 uMMicrotubulesnatural abundance1
1.5 mMTPX2a5-7[U-13C; U-15N],D; 100% back-exchanged2
100 uMMicrotubulesnatural abundance2
25 mMphosphate buffernatural abundance1
25 mMphosphate buffernatural abundance2
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)
125 mM phosphate, 8 mM MgCl2, 1mM DTT, 1mM EGTA Not definedProtonated TPX2/MTs6.8 1 atm276 K
225 mM phosphate, 8 mM MgCl2, 1mM DTT, 1mM EGTA Not definedDeuterated TPX2/MTs (60 kHz MAS)6.8 1 atm283 K
325 mM phosphate, 8 mM MgCl2, 1mM DTT, 1mM EGTA Not definedDeuterated TPX2/MTs (fast MAS2)6.8 1 atm285 K
425 mM phosphate, 8 mM MgCl2, 1mM DTT, 1mM EGTA Not definedDeuterated TPX2/MTs (110 kHz ultrafast MAS)6.8 1 atm288 K
525 mM phosphate, 8 mM MgCl2, 1mM DTT, 1mM EGTA Not definedProtonated TPX2/MTs (higher temp)6.8 1 atm288 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Agilent VARIANAgilentVARIAN6001Bruker Avance III HD console
Bruker AVANCE III HDBrukerAVANCE III HD8502Bruker Avance III HD console; Bruker MAS III unit

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Processing

NMR software
NameDeveloperClassification
TopSpinBruker Biospinprocessing
NMRDrawDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
MestreLab (Mnova / MestReNova / MestReC)MestreLabdata analysis
CcpNmr AnalysisCCPNchemical shift assignment
CcpNmr AnalysisCCPNpeak picking
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorestructure calculation
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorerefinement
TALOSCornilescu, Delaglio and Baxdata analysis
SparkyGoddarddata analysis
RefinementMethod: simulated annealing / Software ordinal: 7
NMR representativeSelection criteria: medoid
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 10

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