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- EMDB-47173: HURP(65-174) bound to GMPCPP-stabilized microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-47173
TitleHURP(65-174) bound to GMPCPP-stabilized microtubule
Map data
Sample
  • Complex: HURP(65-174) bound to GMPCPP-stabilized microtubule
    • Protein or peptide: Disks large-associated protein 5
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Tubulin alpha chain
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
Keywordsmicrotubule / nucleation / spindle / oncogene / CELL CYCLE
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 / COPI-mediated anterograde transport / NOTCH3 Intracellular Domain Regulates Transcription / centrosome localization / 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 / cytosol / cytoplasm
Similarity search - Function
SAPAP family / Guanylate-kinase-associated protein (GKAP) protein / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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 / Disks large-associated protein 5 / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsMa M / Valdez V / Petry S / Zhang R
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47173.png

Downloads & links

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Map

FileDownload / File: emd_47173.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.39 Å/pix.
x 400 pix.
= 556. Å		1.39 Å/pix.
x 400 pix.
= 556. Å		1.39 Å/pix.
x 400 pix.
= 556. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.39 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum0.0 - 1.8258374
Average (Standard dev.)0.009491243 (±0.055514146)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 556.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_47173_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_47173_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_47173_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : HURP(65-174) bound to GMPCPP-stabilized microtubule

EntireName: HURP(65-174) bound to GMPCPP-stabilized microtubule
Components
  • Complex: HURP(65-174) bound to GMPCPP-stabilized microtubule
    • Protein or peptide: Disks large-associated protein 5
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Tubulin alpha chain
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: HURP(65-174) bound to GMPCPP-stabilized microtubule

SupramoleculeName: HURP(65-174) bound to GMPCPP-stabilized microtubule / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Sus scrofa (pig)

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Macromolecule #1: Disks large-associated protein 5

MacromoleculeName: Disks large-associated protein 5 / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 5.682775 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GDQRKQMLQK YKEEKQLQKL KEQREKAKRG IFKVGRYRPD MPCFLL

UniProtKB: Disks large-associated protein 5

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 47.940945 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 QD

UniProtKB: Tubulin beta chain

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Macromolecule #3: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 3 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 48.867195 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 GVDS

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #4: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 4 / Number of copies: 9 / 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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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 18 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 9 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Details: BRB80 (1X): 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 2 / Number real images: 1728 / Average exposure time: 9.0 sec. / Average electron dose: 39.6 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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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

Particle selectionNumber selected: 348514
Startup modelType of model: EMDB MAP
EMDB ID:

7973

Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.4) / Number images used: 2936725
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.4)
Final 3D classificationNumber classes: 100 / Software - Name: cryoSPARC (ver. 3.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: BACKBONE TRACE
Output model

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

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