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- PDB-9eoj: Vertebrate microtubule-capping gamma-tubulin ring complex -

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Basic information

Entry
Database: PDB / ID: 9eoj
TitleVertebrate microtubule-capping gamma-tubulin ring complex
Components
  • (Gamma-tubulin complex ...) x 5
  • Mitotic-spindle organizing protein 1
  • Tubulin gamma-1 chain
KeywordsCELL CYCLE / cytoskeleton / microtubule / microtubule nucleation / complex / template / cap / gamma-tubulin / gamma-tubulin ring complex
Function / homology
Function and homology information


gamma-tubulin complex localization / mitotic spindle microtubule / gamma-tubulin ring complex / polar microtubule / gamma-tubulin complex / microtubule nucleation / gamma-tubulin binding / cytoplasmic microtubule organization / spindle / spindle pole ...gamma-tubulin complex localization / mitotic spindle microtubule / gamma-tubulin ring complex / polar microtubule / gamma-tubulin complex / microtubule nucleation / gamma-tubulin binding / cytoplasmic microtubule organization / spindle / spindle pole / microtubule / centrosome / GTP binding / cytoplasm
Similarity search - Function
Mitotic-spindle organizing protein 1 / Mitotic-spindle organizing gamma-tubulin ring associated / Gamma-tubulin complex component 6, N-terminal / Gamma-tubulin complex component 6 N-terminus / Gamma tubulin / Gamma tubulin complex component, C-terminal / Gamma-tubulin complex component, C-terminal domain superfamily / Gamma tubulin complex component C-terminal / Gamma-tubulin complex component protein / Gamma tubulin complex component protein, N-terminal ...Mitotic-spindle organizing protein 1 / Mitotic-spindle organizing gamma-tubulin ring associated / Gamma-tubulin complex component 6, N-terminal / Gamma-tubulin complex component 6 N-terminus / Gamma tubulin / Gamma tubulin complex component, C-terminal / Gamma-tubulin complex component, C-terminal domain superfamily / Gamma tubulin complex component C-terminal / Gamma-tubulin complex component protein / Gamma tubulin complex component protein, N-terminal / Gamma tubulin complex component N-terminal / 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
Gamma-tubulin complex component / Gamma-tubulin complex component / Gamma-tubulin complex component 6 / Gamma-tubulin complex component 3 homolog / Tubulin gamma-1 chain / Mitotic-spindle organizing protein 1 / Gamma-tubulin complex component
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 17 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 195/4-4 Germany
Other private
German Research Foundation (DFG)INST 35/1503-1 FUGG Germany
German Research Foundation (DFG)INST 35/1134-1 FUGG Germany
German Research Foundation (DFG)INST 35/1314-1 FUGG Germany
CitationJournal: 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 1.0Apr 17, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Mitotic-spindle organizing protein 1
D: Mitotic-spindle organizing protein 1
Q: Gamma-tubulin complex component 3 homolog
R: Gamma-tubulin complex component
S: Gamma-tubulin complex component 6
T: Tubulin gamma-1 chain
U: Gamma-tubulin complex component
V: Gamma-tubulin complex component
W: Gamma-tubulin complex component
Y: Gamma-tubulin complex component
Z: Gamma-tubulin complex component
a: Gamma-tubulin complex component
b: Gamma-tubulin complex component
c: Gamma-tubulin complex component 3 homolog
d: Gamma-tubulin complex component 3 homolog
e: Gamma-tubulin complex component 3 homolog
f: Gamma-tubulin complex component 3 homolog
h: Tubulin gamma-1 chain
i: Tubulin gamma-1 chain
k: Tubulin gamma-1 chain
l: Tubulin gamma-1 chain
m: Tubulin gamma-1 chain
n: Tubulin gamma-1 chain
o: Tubulin gamma-1 chain
p: Tubulin gamma-1 chain
q: Tubulin gamma-1 chain
r: Tubulin gamma-1 chain
s: Tubulin gamma-1 chain
t: Tubulin gamma-1 chain
w: Tubulin gamma-1 chain


Theoretical massNumber of molelcules
Total (without water)2,231,85530
Polymers2,231,85530
Non-polymers00
Water0
1


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

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Components

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Protein , 2 types, 16 molecules BDThiklmnopqrstw

#1: Protein Mitotic-spindle organizing protein 1 / Mitotic-spindle organizing protein associated with a ring of gamma-tubulin 1


Mass: 7749.854 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q5U4M5
#5: Protein
Tubulin gamma-1 chain / Gamma-1-tubulin / xGAM


Mass: 51226.719 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: P23330

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Gamma-tubulin complex ... , 5 types, 14 molecules QcdefRYZabSUVW

#2: Protein
Gamma-tubulin complex component 3 homolog / Gamma-ring complex protein 109 / Xgrip109 / x109p


Mass: 103789.352 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: O73787
#3: Protein
Gamma-tubulin complex component


Mass: 103468.312 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A8J0T6B8
#4: Protein Gamma-tubulin complex component 6


Mass: 193069.141 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A974HT83
#6: Protein Gamma-tubulin complex component


Mass: 117577.633 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8HGZ5
#7: Protein Gamma-tubulin complex component


Mass: 76122.961 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q642S3

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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: Vertebrate microtubule-capping gamma-tubulin ring complex
Type: COMPLEX
Details: Vertebrate microtubule-capping gamma-tubulin ring complex induced through the RanGTP spindle assembly pathway and isolated from Xenopus laevis egg extract
Entity ID: all / 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 FIELDBright-field microscopy / Nominal magnification: 33000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
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
7UCSF Chimera1.14model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8497 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16TF9

6tf9

16TF91PDBexperimental model
21AlphaFoldin silico model

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