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- PDB-7qja: Structure of recombinant human gamma-Tubulin Ring Complex 12-spok... -

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Basic information

Entry
Database: PDB / ID: 7qja
TitleStructure of recombinant human gamma-Tubulin Ring Complex 12-spoked assembly intermediate (spokes 1-12, homogeneous dataset)
Components
  • (Gamma-tubulin complex component ...) x 5
  • Mitotic-spindle organizing protein 1
  • Tubulin gamma-1 chain
  • actin, cytoplasmic 1
KeywordsCYTOSOLIC PROTEIN / Assembly / Intermediate / Complex
Function / homology
Function and homology information


microtubule nucleation by interphase microtubule organizing center / gamma-tubulin complex localization / equatorial microtubule organizing center / mitotic spindle microtubule / gamma-tubulin ring complex / interphase microtubule organizing center / polar microtubule / gamma-tubulin complex / meiotic spindle organization / microtubule nucleation ...microtubule nucleation by interphase microtubule organizing center / gamma-tubulin complex localization / equatorial microtubule organizing center / mitotic spindle microtubule / gamma-tubulin ring complex / interphase microtubule organizing center / polar microtubule / gamma-tubulin complex / meiotic spindle organization / microtubule nucleation / non-motile cilium / gamma-tubulin binding / microtubule organizing center / pericentriolar material / single fertilization / cell leading edge / mitotic sister chromatid segregation / mitotic spindle assembly / spindle assembly / cytoplasmic microtubule / 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 / meiotic cell cycle / condensed nuclear chromosome / brain development / neuron migration / structural constituent of cytoskeleton / spindle / microtubule cytoskeleton organization / recycling endosome / spindle pole / microtubule cytoskeleton / Regulation of PLK1 Activity at G2/M Transition / apical part of cell / mitotic cell cycle / microtubule binding / protein-containing complex assembly / microtubule / hydrolase activity / neuron projection / centrosome / GTP binding / structural molecule activity / nucleoplasm / membrane / identical protein binding / 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 / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / 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 / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Actin, cytoplasmic 1 / 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: 9.2 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

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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
e: actin, cytoplasmic 1
l: Gamma-tubulin complex component 5
B: Gamma-tubulin complex component 3
C: Gamma-tubulin complex component 2
D: Gamma-tubulin complex component 3
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
b: Mitotic-spindle organizing protein 1
g: Mitotic-spindle organizing protein 1
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
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
i: Mitotic-spindle organizing protein 1
k: Mitotic-spindle organizing protein 1
m: Mitotic-spindle organizing protein 1
a: Gamma-tubulin complex component 3
f: Gamma-tubulin complex component 3
h: Gamma-tubulin complex component 3
A: Gamma-tubulin complex component 2
j: Gamma-tubulin complex component 3
d: Mitotic-spindle organizing protein 1
c: Gamma-tubulin complex component 6


Theoretical massNumber of molelcules
Total (without water)2,738,54137
Polymers2,738,54137
Non-polymers00
Water0
1


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

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Components

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Protein , 3 types, 19 molecules ebgikmdOPQRSTUVWXYZ

#1: Protein actin, cytoplasmic 1 /


Mass: 41782.660 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTB
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: A0A6I9HGD1
#7: 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.: 6
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
#8: Protein
Tubulin gamma-1 chain / Gamma-1-tubulin / Gamma-tubulin complex component 1 / GCP-1


Mass: 51227.770 Da / Num. of mol.: 12
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

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Gamma-tubulin complex component ... , 5 types, 18 molecules lJBDFHafhjCEGAIKLc

#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.: 8
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
#4: 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.: 4
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
#5: 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
#6: Protein Gamma-tubulin complex component 6 / GCP-6


Mass: 200733.641 Da / Num. of mol.: 2
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: Recombinant human Gamma-Tubulin Ring Complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 2.0 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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm
Image recordingElectron dose: 35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6127

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.4particle selection
2EPU2.6image acquisition
4Gctf1.06CTF correction
7NAMDmodel fitting
8VMDmodel fitting
10PHENIXmodel refinement
11RELION3.1initial Euler assignment
12RELION3final Euler assignment
13RELION3.1classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 436887
3D reconstructionResolution: 9.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 5376 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
17AS4

7as4

A1
27AS4

7as4

B1
37AS4

7as4

K1
47AS4

7as4

O1
57AS4

7as4

71
66V6S

6v6s

J1
76V6S

6v6s

L1
86L81

6l81

1
96X0U

6x0u

1

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