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- PDB-9g40: Structure of the Position 7 CMG-decorated gamma-Tubulin Ring Comp... -

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Basic information

Entry
Database: PDB / ID: 9g40
TitleStructure of the Position 7 CMG-decorated gamma-Tubulin Ring Complex from Pig Brain
Components
  • CDK5 regulatory subunit-associated protein 2
  • Gamma-tubulin complex component
  • Gamma-tubulin complex component 3
  • Mitotic-spindle organizing protein 2A isoform X4
KeywordsSTRUCTURAL PROTEIN / Tubulin Complex
Function / homology
Function and homology information


microtubule organizing center organization / Recruitment of mitotic centrosome proteins and complexes / negative regulation of centriole replication / equatorial microtubule organizing center / regulation of mitotic cell cycle spindle assembly checkpoint / polar microtubule / microtubule plus-end / gamma-tubulin complex / microtubule nucleation / microtubule bundle formation ...microtubule organizing center organization / Recruitment of mitotic centrosome proteins and complexes / negative regulation of centriole replication / equatorial microtubule organizing center / regulation of mitotic cell cycle spindle assembly checkpoint / polar microtubule / microtubule plus-end / gamma-tubulin complex / microtubule nucleation / microtubule bundle formation / gamma-tubulin binding / centrosome cycle / Recruitment of NuMA to mitotic centrosomes / regulation of neuron differentiation / pericentriolar material / mitotic spindle pole / establishment of mitotic spindle orientation / centriole replication / negative regulation of neuron differentiation / spindle assembly / cytoplasmic microtubule organization / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Recruitment of NuMA to mitotic centrosomes / positive regulation of microtubule polymerization / Anchoring of the basal body to the plasma membrane / centriole / AURKA Activation by TPX2 / tubulin binding / neurogenesis / meiotic cell cycle / chromosome segregation / neuron migration / brain development / microtubule cytoskeleton organization / spindle pole / spindle / Regulation of PLK1 Activity at G2/M Transition / cell junction / mitotic cell cycle / microtubule binding / microtubule / cytoskeleton / calmodulin binding / transcription cis-regulatory region binding / centrosome / protein-containing complex binding / protein kinase binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / Golgi apparatus / extracellular exosome / nucleoplasm / cytosol / cytoplasm
Similarity search - Function
CDK5 regulatory subunit-associated protein 2 / MOZART2 family / Mitotic-spindle organizing gamma-tubulin ring associated / Centrosomin, N-terminal motif 1 / Centrosomin N-terminal motif 1 / 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
Similarity search - Domain/homology
Gamma-tubulin complex component / Mitotic-spindle organizing protein 2A isoform X4 / Gamma-tubulin complex component 3 / CDK5 regulatory subunit-associated protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsMunoz-Hernandez, H. / Krutyholowa, R. / Wieczorek, M.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science FoundationTMSGI3_211309 Switzerland
Swiss National Science Foundation310030_208120 Switzerland
CitationJournal: Dev Cell / Year: 2024
Title: Partial closure of the γ-tubulin ring complex by CDK5RAP2 activates microtubule nucleation.
Authors: Yixin Xu / Hugo Muñoz-Hernández / Rościsław Krutyhołowa / Florina Marxer / Ferdane Cetin / Michal Wieczorek /
Abstract: Microtubule nucleation is templated by the γ-tubulin ring complex (γ-TuRC), but its structure deviates from the geometry of α-/β-tubulin in the microtubule, explaining the complex's poor ...Microtubule nucleation is templated by the γ-tubulin ring complex (γ-TuRC), but its structure deviates from the geometry of α-/β-tubulin in the microtubule, explaining the complex's poor nucleating activity. Several proteins may activate the γ-TuRC, but the mechanisms underlying activation are not known. Here, we determined the structure of the porcine γ-TuRC purified using CDK5RAP2's centrosomin motif 1 (CM1). We identified an unexpected conformation of the γ-TuRC bound to multiple protein modules containing MZT2, GCP2, and CDK5RAP2, resulting in a long-range constriction of the γ-tubulin ring that brings it in closer agreement with the 13-protofilament microtubule. Additional CDK5RAP2 promoted γ-TuRC decoration and stimulated the microtubule-nucleating activities of the porcine γ-TuRC and a reconstituted, CM1-free human complex in single-molecule assays. Our results provide a structural mechanism for the control of microtubule nucleation by CM1 proteins and identify conformational transitions in the γ-TuRC that prime it for microtubule nucleation.
History
DepositionJul 12, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin / pdbx_entry_details
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
F: Gamma-tubulin complex component 3
G: Gamma-tubulin complex component
X: Mitotic-spindle organizing protein 2A isoform X4
u: CDK5 regulatory subunit-associated protein 2
v: CDK5 regulatory subunit-associated protein 2


Theoretical massNumber of molelcules
Total (without water)652,3915
Polymers652,3915
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Gamma-tubulin complex component 3 / Tubulin gamma complex associated protein 3


Mass: 103172.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: SsGCP3 / Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: F1RN46
#2: Protein Gamma-tubulin complex component


Mass: 102609.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: A0A480VJI0
#3: Protein Mitotic-spindle organizing protein 2A isoform X4


Mass: 15920.321 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: F1RK97
#4: Protein CDK5 regulatory subunit-associated protein 2 / CDK5 activator-binding protein C48 / Centrosome-associated protein 215


Mass: 215344.219 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDK5RAP2, CEP215, KIAA1633 / Details (production host): (Kan) / Production host: Escherichia coli (E. coli) / Strain (production host): pRARE / References: UniProt: Q96SN8
Has protein modificationN

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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: Gamma-Tubulin Ring Complex in native pig brain / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Sus scrofa (pig)
Buffer solutionpH: 7.5
Details: Listed in "g-TuRC cryo-EM sample preparation" section.
Buffer component
IDConc.NameBuffer-ID
140 mMHEPES1
2150 mMNaCl1
31 mMMgCl21
41 mMEGTA1
52 mM2-mercaptoethanol1
60.01 % v/vIGEPAL1
70.1 mMGTP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2700 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93207 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212695
ELECTRON MICROSCOPYf_angle_d0.42917136
ELECTRON MICROSCOPYf_dihedral_angle_d11.6454763
ELECTRON MICROSCOPYf_chiral_restr0.0321914
ELECTRON MICROSCOPYf_plane_restr0.0032188

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