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- PDB-9eok: Minus end of the vertebrate gamma-tubulin ring complex-capped mic... -

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Basic information

Entry
Database: PDB / ID: 9eok
TitleMinus end of the vertebrate gamma-tubulin ring complex-capped microtubule
Components
  • Tubulin alpha chain
  • Tubulin beta-4 chain
KeywordsCELL CYCLE / cytoskeleton / microtubule / microtubule nucleation / complex / template / cap / gamma-tubulin / gamma-tubulin ring complex
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
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-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
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin beta-4 chain / Tubulin alpha chain
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 23 Å
AuthorsVermeulen, B.J.A. / Pfeffer, S.
Funding support Germany, 6items
OrganizationGrant numberCountry
German Research Foundation (DFG)PF 963/1-4 Germany
German Research Foundation (DFG)Schi 295/4-4 Germany
Other private
German Research Foundation (DFG)INST 35/1314-1 FUGG Germany
German Research Foundation (DFG)INST 35/1503-1 FUGG Germany
German Research Foundation (DFG)INST 35/1134-1 FUGG Germany
Citation
Journal: EMBO J / Year: 2024
Title: γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end.
Authors: Bram Ja Vermeulen / Anna Böhler / Qi Gao / Annett Neuner / Erik Župa / Zhenzhen Chu / Martin Würtz / Ursula Jäkle / Oliver J Gruss / Stefan Pfeffer / Elmar Schiebel /
Abstract: The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure ...The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.
#1: Journal: EMBO J / Year: 2024
Title: γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end.
Authors: Bram Ja Vermeulen / Anna Böhler / Qi Gao / Annett Neuner / Erik Župa / Zhenzhen Chu / Martin Würtz / Ursula Jäkle / Oliver J Gruss / Stefan Pfeffer / Elmar Schiebel /
Abstract: The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure ...The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.
History
DepositionMar 15, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tubulin alpha chain
B: Tubulin beta-4 chain
C: Tubulin alpha chain
D: Tubulin alpha chain
E: Tubulin alpha chain
F: Tubulin beta-4 chain
G: Tubulin alpha chain
H: Tubulin alpha chain
I: Tubulin alpha chain
J: Tubulin alpha chain
K: Tubulin alpha chain
L: Tubulin alpha chain
M: Tubulin alpha chain
N: Tubulin alpha chain
O: Tubulin alpha chain
P: Tubulin alpha chain
Q: Tubulin beta-4 chain
R: Tubulin beta-4 chain
S: Tubulin beta-4 chain
T: Tubulin beta-4 chain
U: Tubulin beta-4 chain
V: Tubulin beta-4 chain
W: Tubulin beta-4 chain
X: Tubulin beta-4 chain
Y: Tubulin beta-4 chain
Z: Tubulin beta-4 chain
a: Tubulin beta-4 chain
b: Tubulin beta-4 chain
c: Tubulin alpha chain
d: Tubulin alpha chain
e: Tubulin alpha chain
f: Tubulin alpha chain
g: Tubulin alpha chain
h: Tubulin alpha chain
i: Tubulin alpha chain
j: Tubulin alpha chain
k: Tubulin alpha chain
o: Tubulin beta-4 chain
p: Tubulin beta-4 chain
q: Tubulin beta-4 chain
r: Tubulin beta-4 chain
s: Tubulin beta-4 chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,137,397126
Polymers2,100,15942
Non-polymers37,23784
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 42 molecules ACDEGHIJKLMNOPcdefghijkBFQRSTU...

#1: Protein ...
Tubulin alpha chain


Mass: 50120.297 Da / Num. of mol.: 23 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q5U4V6
#2: Protein
Tubulin beta-4 chain


Mass: 49862.770 Da / Num. of mol.: 19 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: P30883

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Non-polymers , 4 types, 84 molecules

#3: Chemical...
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 23 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#4: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 23 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 19 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#6: Chemical
ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 19 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
Type: COMPLEX
Details: Minus end of the vertebrate gamma-tubulin ring complex-capped microtubule induced through the RanGTP spindle assembly pathway and isolated from Xenopus laevis egg extract
Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 33000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 43 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionManual picking
2EPUimage acquisition
4Gctf1.06CTF correction
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8497 / Symmetry type: POINT

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