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- PDB-7qj4: Structure of recombinant human gamma-Tubulin Ring Complex 10-spok... -

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Basic information

Entry
Database: PDB / ID: 7qj4
TitleStructure of recombinant human gamma-Tubulin Ring Complex 10-spoked assembly intermediate (spokes 5-14)
Components
  • (Gamma-tubulin complex component ...) x 5
  • Mitotic-spindle organizing protein 1
  • Tubulin gamma-1 chain
KeywordsCYTOSOLIC PROTEIN / Intermediate / Assembly / Complex
Function / homology
Function and homology information


microtubule nucleation by interphase microtubule organizing center / gamma-tubulin complex localization / equatorial microtubule organizing center / gamma-tubulin ring complex / interphase microtubule organizing center / polar microtubule / mitotic spindle microtubule / meiotic spindle organization / gamma-tubulin complex / microtubule nucleation ...microtubule nucleation by interphase microtubule organizing center / gamma-tubulin complex localization / equatorial microtubule organizing center / gamma-tubulin ring complex / interphase microtubule organizing center / polar microtubule / mitotic spindle microtubule / meiotic spindle organization / gamma-tubulin complex / microtubule nucleation / non-motile cilium / gamma-tubulin binding / microtubule organizing center / pericentriolar material / cytoplasmic microtubule / cell leading edge / mitotic sister chromatid segregation / mitotic spindle assembly / single fertilization / 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 / Anchoring of the basal body to the plasma membrane / centriole / AURKA Activation by TPX2 / mitotic spindle organization / ciliary basal body / condensed nuclear chromosome / meiotic cell cycle / neuron migration / brain development / recycling endosome / structural constituent of cytoskeleton / microtubule cytoskeleton organization / spindle pole / spindle / microtubule cytoskeleton / Regulation of PLK1 Activity at G2/M Transition / apical part of cell / mitotic cell cycle / microtubule binding / protein-containing complex assembly / microtubule / neuron projection / centrosome / GTP binding / structural molecule activity / nucleoplasm / identical protein binding / membrane / nucleus / cytosol / 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
Tubulin gamma-1 chain / Mitotic-spindle organizing protein 1 / Gamma-tubulin complex component 3 / Gamma-tubulin complex component 6 / Gamma-tubulin complex component 5 / Gamma-tubulin complex component 2 / Gamma-tubulin complex component 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9 Å
AuthorsZupa, E. / Pfeffer, S.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)PF 963/1-4 Germany
German Research Foundation (DFG)Schi 295/4-4 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Modular assembly of the principal microtubule nucleator γ-TuRC.
Authors: Martin Würtz / Erik Zupa / Enrico S Atorino / Annett Neuner / Anna Böhler / Ariani S Rahadian / Bram J A Vermeulen / Giulia Tonon / Sebastian Eustermann / Elmar Schiebel / Stefan Pfeffer /
Abstract: The gamma-tubulin ring complex (γ-TuRC) is the principal microtubule nucleation template in vertebrates. Recent cryo-EM reconstructions visualized the intricate quaternary structure of the γ-TuRC, ...The gamma-tubulin ring complex (γ-TuRC) is the principal microtubule nucleation template in vertebrates. Recent cryo-EM reconstructions visualized the intricate quaternary structure of the γ-TuRC, containing more than thirty subunits, raising fundamental questions about γ-TuRC assembly and the role of actin as an integral part of the complex. Here, we reveal the structural mechanism underlying modular γ-TuRC assembly and identify a functional role of actin in microtubule nucleation. During γ-TuRC assembly, a GCP6-stabilized core comprising GCP2-3-4-5-4-6 is expanded by stepwise recruitment, selective stabilization and conformational locking of four pre-formed GCP2-GCP3 units. Formation of the lumenal bridge specifies incorporation of the terminal GCP2-GCP3 unit and thereby leads to closure of the γ-TuRC ring in a left-handed spiral configuration. Actin incorporation into the complex is not relevant for γ-TuRC assembly and structural integrity, but determines γ-TuRC geometry and is required for efficient microtubule nucleation and mitotic chromosome alignment in vivo.
History
DepositionDec 16, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
1: Tubulin gamma-1 chain
2: Tubulin gamma-1 chain
l: Gamma-tubulin complex component 5
n: Gamma-tubulin complex component 3
o: Mitotic-spindle organizing protein 1
m: Mitotic-spindle organizing protein 1
E: Gamma-tubulin complex component 2
F: Gamma-tubulin complex component 3
G: Gamma-tubulin complex component 2
H: Gamma-tubulin complex component 3
I: Gamma-tubulin complex component 4
J: Gamma-tubulin complex component 5
K: Gamma-tubulin complex component 4
L: Gamma-tubulin complex component 6
M: Gamma-tubulin complex component 2
N: Gamma-tubulin complex component 3
k: Mitotic-spindle organizing protein 1
b: Mitotic-spindle organizing protein 1
j: Gamma-tubulin complex component 3
a: Gamma-tubulin complex component 3
S: Tubulin gamma-1 chain
T: Tubulin gamma-1 chain
U: Tubulin gamma-1 chain
V: Tubulin gamma-1 chain
W: Tubulin gamma-1 chain
X: Tubulin gamma-1 chain
Y: Tubulin gamma-1 chain
Z: Tubulin gamma-1 chain


Theoretical massNumber of molelcules
Total (without water)2,066,51128
Polymers2,066,51128
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 14 molecules 12STUVWXYZomkb

#1: Protein
Tubulin gamma-1 chain / Gamma-1-tubulin / Gamma-tubulin complex component 1 / GCP-1


Mass: 51227.770 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBG1, TUBG
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: P23258
#4: Protein
Mitotic-spindle organizing protein 1 / Mitotic-spindle organizing protein associated with a ring of gamma-tubulin 1


Mass: 8485.724 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MZT1, C13orf37, MOZART1
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: Q08AG7

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Gamma-tubulin complex component ... , 5 types, 14 molecules lJnFHNjaEGMIKL

#2: Protein Gamma-tubulin complex component 5 / GCP-5


Mass: 118467.547 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBGCP5, GCP5, KIAA1899
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: Q96RT8
#3: Protein
Gamma-tubulin complex component 3 / hGCP3 / Gamma-ring complex protein 104 kDa / hGrip104 / Spindle pole body protein Spc98 homolog / hSpc98


Mass: 103710.102 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBGCP3, GCP3
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: Q96CW5
#5: Protein Gamma-tubulin complex component 2 / hGCP2 / Gamma-ring complex protein 103 kDa / hGrip103 / Spindle pole body protein Spc97 homolog / hSpc97


Mass: 102666.953 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBGCP2, GCP2
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: Q9BSJ2
#6: Protein Gamma-tubulin complex component 4 / hGCP4 / Gamma-ring complex protein 76 kDa / hGrip76


Mass: 76179.969 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBGCP4, 76P, GCP4
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: Q9UGJ1
#7: Protein Gamma-tubulin complex component 6 / GCP-6


Mass: 200733.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBGCP6, GCP6, KIAA1669
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: Q96RT7

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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: 10-spoked assembly intermediate of the gamma-Tubulin Ring Complex (spokes 5-14)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.6 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Buffer solutionpH: 7.5
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
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 2000 nm
Image recordingElectron dose: 35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 6127

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Processing

SoftwareName: PHENIX / Version: dev_3699: / Classification: refinement
EM software
IDNameVersionCategory
1Topaz0.2.4particle selection
2EPU2.6image acquisition
4Gctf1.06CTF correction
7NAMDmodel fitting
8VMDmodel fitting
10RELION3.1initial Euler assignment
11RELION3final Euler assignment
12RELION3.1classification
13RELION33D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 436887
3D reconstructionResolution: 9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6097 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
17AS4

7as4

A7AS41
27AS4

7as4

B7AS41
37AS4

7as4

K7AS41
46V6S

6v6s

J6V6S2
56V6S

6v6s

L6V6S2
67AS4

7as4

O7AS41
77AS4

7as4

K7AS41
86X0U

6x0u

6X0U3
96L81

6l81

6L814
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01989138
ELECTRON MICROSCOPYf_angle_d1.268120619
ELECTRON MICROSCOPYf_dihedral_angle_d22.39712484
ELECTRON MICROSCOPYf_chiral_restr0.06713570
ELECTRON MICROSCOPYf_plane_restr0.00815469

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