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- PDB-9duq: HURP(65-174) bound to GMPCPP-stabilized microtubule -

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Basic information

Entry
Database: PDB / ID: 9duq
TitleHURP(65-174) bound to GMPCPP-stabilized microtubule
Components
  • Disks large-associated protein 5
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsCELL CYCLE / microtubule / nucleation / spindle / oncogene
Function / homology
Function and homology information


mitotic chromosome movement towards spindle pole / signaling / kinetochore assembly / positive regulation of mitotic metaphase/anaphase transition / spindle pole centrosome / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport ...mitotic chromosome movement towards spindle pole / signaling / kinetochore assembly / positive regulation of mitotic metaphase/anaphase transition / spindle pole centrosome / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / EML4 and NUDC in mitotic spindle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / 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 / NOTCH3 Intracellular Domain Regulates Transcription / centrosome localization / COPI-mediated anterograde transport / regulation of mitotic cell cycle / mitotic spindle organization / chromosome segregation / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic spindle / mitotic cell cycle / microtubule cytoskeleton / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / GTPase activity / GTP binding / metal ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
SAPAP family / Guanylate-kinase-associated protein (GKAP) 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 ...SAPAP family / Guanylate-kinase-associated protein (GKAP) 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
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta chain / Disks large-associated protein 5 / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsMa, M. / Valdez, V. / Petry, S. / Zhang, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM138854 United States
CitationJournal: Nat Commun / Year: 2024
Title: HURP facilitates spindle assembly by stabilizing microtubules and working synergistically with TPX2.
Authors: Venecia Alexandria Valdez / Meisheng Ma / Bernardo Gouveia / Rui Zhang / Sabine Petry /
Abstract: In vertebrate spindles, most microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein ...In vertebrate spindles, most microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from γ-TuRC. This function is shifted to promote branching microtubule nucleation through enhanced localization to TPX2 condensates, which form the core of the branch site on microtubules. Lastly, we provide a high-resolution cryo-EM structure of HURP on the microtubule, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.
History
DepositionOct 3, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
r: Disks large-associated protein 5
B: Tubulin beta chain
A: Tubulin alpha chain
D: Tubulin beta chain
C: Tubulin alpha chain
F: Tubulin beta chain
E: Tubulin alpha chain
H: Tubulin beta chain
G: Tubulin alpha chain
J: Tubulin beta chain
I: Tubulin alpha chain
L: Tubulin beta chain
K: Tubulin alpha chain
N: Tubulin beta chain
M: Tubulin alpha chain
P: Tubulin beta chain
O: Tubulin alpha chain
R: Tubulin beta chain
Q: Tubulin alpha chain
s: Disks large-associated protein 5
t: Disks large-associated protein 5
u: Disks large-associated protein 5
v: Disks large-associated protein 5
w: Disks large-associated protein 5
x: Disks large-associated protein 5
y: Disks large-associated protein 5
z: Disks large-associated protein 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)932,25563
Polymers922,41827
Non-polymers9,83736
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 1 types, 9 molecules rstuvwxyz

#1: Protein/peptide
Disks large-associated protein 5 / DAP-5 / Discs large homolog 7 / Disks large-associated protein DLG7 / Hepatoma up-regulated protein / HURP


Mass: 5682.775 Da / Num. of mol.: 9 / Fragment: UNP residues 87-132
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DLGAP5, DLG7, KIAA0008 / Production host: Escherichia coli (E. coli) / References: UniProt: Q15398

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Protein , 2 types, 18 molecules BDFHJLNPRACEGIKMOQ

#2: Protein
Tubulin beta chain / Beta-tubulin


Mass: 47940.945 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554
#3: Protein
Tubulin alpha chain


Mass: 48867.195 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: Q2XVP4

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Non-polymers , 3 types, 36 molecules

#4: Chemical
ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-CPP, energy-carrying molecule analogue*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Mg
#6: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: HURP(65-174) bound to GMPCPP-stabilized microtubule / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Sus scrofa (pig)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8
Details: BRB80 (1X): 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 0.01 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 39.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1728
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.4particle selection
2EPUimage acquisition
4cryoSPARC3.4CTF correction
7Cootmodel fitting
9cryoSPARC3.4initial Euler assignment
10cryoSPARC3.4final Euler assignment
11cryoSPARC3.4classification
12cryoSPARC3.43D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 348514
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2936725 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: RECIPROCAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00465970
ELECTRON MICROSCOPYf_angle_d0.63689469
ELECTRON MICROSCOPYf_dihedral_angle_d10.4029000
ELECTRON MICROSCOPYf_chiral_restr0.0489711
ELECTRON MICROSCOPYf_plane_restr0.00511637

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