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- PDB-6tf9: Structure of the vertebrate gamma-Tubulin Ring Complex -

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Basic information

Entry
Database: PDB / ID: 6tf9
TitleStructure of the vertebrate gamma-Tubulin Ring Complex
Components
  • (Belt helices ...) x 4
  • (Gamma-tubulin complex ...) x 4
  • Actin, cytoplasmic 1
  • Belt helix 16
  • Belt helix 17
  • Belt helix 5
  • Belt helix 6
  • Belt helix 7
  • Gamma tubulin ring protein
  • Helix 1
  • Tubulin gamma-1 chain
KeywordsCELL CYCLE / gamma-Tubulin Ring Complex / microtubule nucleation
Function / homology
Function and homology information


mitotic spindle microtubule / polar microtubule / gamma-tubulin complex / dense body / microtubule nucleation / gamma-tubulin binding / microtubule organizing center / cytoplasmic microtubule organization / spindle pole / actin cytoskeleton ...mitotic spindle microtubule / polar microtubule / gamma-tubulin complex / dense body / microtubule nucleation / gamma-tubulin binding / microtubule organizing center / cytoplasmic microtubule organization / spindle pole / actin cytoskeleton / microtubule / cytoskeleton / focal adhesion / centrosome / GTP binding / ATP binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
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 ...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
Gamma-tubulin complex component / Gamma-tubulin complex component 3 homolog / Actin, cytoplasmic 1 / Tubulin gamma-1 chain / Gamma-tubulin complex component / Gamma tubulin ring protein
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsZupa, E. / Pfeffer, S.
CitationJournal: Nature / Year: 2020
Title: Insights into the assembly and activation of the microtubule nucleator γ-TuRC.
Authors: Peng Liu / Erik Zupa / Annett Neuner / Anna Böhler / Justus Loerke / Dirk Flemming / Thomas Ruppert / Till Rudack / Christoph Peter / Christian Spahn / Oliver J Gruss / Stefan Pfeffer / Elmar Schiebel /
Abstract: Microtubules are dynamic polymers of α- and β-tubulin and have crucial roles in cell signalling, cell migration, intracellular transport and chromosome segregation. They assemble de novo from αβ- ...Microtubules are dynamic polymers of α- and β-tubulin and have crucial roles in cell signalling, cell migration, intracellular transport and chromosome segregation. They assemble de novo from αβ-tubulin dimers in an essential process termed microtubule nucleation. Complexes that contain the protein γ-tubulin serve as structural templates for the microtubule nucleation reaction. In vertebrates, microtubules are nucleated by the 2.2-megadalton γ-tubulin ring complex (γ-TuRC), which comprises γ-tubulin, five related γ-tubulin complex proteins (GCP2-GCP6) and additional factors. GCP6 is unique among the GCP proteins because it carries an extended insertion domain of unknown function. Our understanding of microtubule formation in cells and tissues is limited by a lack of high-resolution structural information on the γ-TuRC. Here we present the cryo-electron microscopy structure of γ-TuRC from Xenopus laevis at 4.8 Å global resolution, and identify a 14-spoked arrangement of GCP proteins and γ-tubulins in a partially flexible open left-handed spiral with a uniform sequence of GCP variants. By forming specific interactions with other GCP proteins, the GCP6-specific insertion domain acts as a scaffold for the assembly of the γ-TuRC. Unexpectedly, we identify actin as a bona fide structural component of the γ-TuRC with functional relevance in microtubule nucleation. The spiral geometry of γ-TuRC is suboptimal for microtubule nucleation and a controlled conformational rearrangement of the γ-TuRC is required for its activation. Collectively, our cryo-electron microscopy reconstructions provide detailed insights into the molecular organization, assembly and activation mechanism of vertebrate γ-TuRC, and will serve as a framework for the mechanistic understanding of fundamental biological processes associated with microtubule nucleation, such as meiotic and mitotic spindle formation and centriole biogenesis.
History
DepositionNov 13, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 11, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Mar 4, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-10491
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Structure viewerMolecule:
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Assembly

Deposited unit
AP1: Helix 1
CP1: Belt helices 1,2,3,4
DP1: Belt helix 5
EP1: Belt helix 6
FP1: Belt helix 7
GP1: Belt helices 8,9,10
HP1: Belt helices 1,2,3,4
IP1: Belt helices 1,2,3,4
JP1: Belt helices 11,12
KP1: Belt helices 13,14,15 and Helix 2
LP1: Belt helices 8,9,10
MP1: Belt helix 16
NP1: Belt helices 8,9,10
OP1: Belt helices 13,14,15 and Helix 2
PP1: Belt helices 13,14,15 and Helix 2
QP1: Gamma-tubulin complex component 3 homolog
RP1: Gamma-tubulin complex component 2
SP1: Gamma tubulin ring protein
TP1: Tubulin gamma-1 chain
UP1: Gamma-tubulin complex component
VP1: Gamma-tubulin complex component
WP1: Gamma-tubulin complex component
XP1: Belt helices 1,2,3,4
YP1: Gamma-tubulin complex component 2
ZP1: Gamma-tubulin complex component 2
aP1: Gamma-tubulin complex component 2
bP1: Gamma-tubulin complex component 2
cP1: Gamma-tubulin complex component 3 homolog
dP1: Gamma-tubulin complex component 3 homolog
eP1: Gamma-tubulin complex component 3 homolog
fP1: Gamma-tubulin complex component 3 homolog
gP1: Belt helix 17
hP1: Tubulin gamma-1 chain
iP1: Tubulin gamma-1 chain
jP1: Actin, cytoplasmic 1
kP1: Tubulin gamma-1 chain
lP1: Tubulin gamma-1 chain
mP1: Tubulin gamma-1 chain
nP1: Tubulin gamma-1 chain
oP1: Tubulin gamma-1 chain
pP1: Tubulin gamma-1 chain
qP1: Tubulin gamma-1 chain
rP1: Tubulin gamma-1 chain
sP1: Tubulin gamma-1 chain
tP1: Tubulin gamma-1 chain
uP1: Belt helices 13,14,15 and Helix 2
vP1: Belt helices 11,12
wP1: Tubulin gamma-1 chain


Theoretical massNumber of molelcules
Total (without water)2,273,11848
Polymers2,273,11848
Non-polymers00
Water0
1


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

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Components

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Protein/peptide , 6 types, 6 molecules AP1DP1EP1FP1MP1gP1

#1: Protein/peptide Helix 1 /


Mass: 2079.272 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#3: Protein/peptide Belt helix 5


Mass: 2221.427 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#4: Protein/peptide Belt helix 6


Mass: 1652.805 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#5: Protein/peptide Belt helix 7


Mass: 1297.416 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#9: Protein/peptide Belt helix 16


Mass: 1226.338 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#16: Protein/peptide Belt helix 17


Mass: 1368.494 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)

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Belt helices ... , 4 types, 13 molecules CP1HP1IP1XP1GP1LP1NP1JP1vP1KP1OP1PP1uP1

#2: Protein/peptide
Belt helices 1,2,3,4


Mass: 942.027 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#6: Protein/peptide Belt helices 8,9,10


Mass: 1084.182 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#7: Protein/peptide Belt helices 11,12


Mass: 1013.105 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#8: Protein/peptide
Belt helices 13,14,15 and Helix 2


Mass: 1155.260 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)

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Gamma-tubulin complex ... , 4 types, 13 molecules QP1cP1dP1eP1fP1RP1YP1ZP1aP1bP1UP1VP1WP1

#10: 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
#11: Protein
Gamma-tubulin complex component 2


Mass: 103468.312 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog)
#14: 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
#15: 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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Protein , 3 types, 16 molecules SP1TP1hP1iP1kP1lP1mP1nP1oP1pP1qP1rP1sP1tP1wP1jP1

#12: Protein Gamma tubulin ring protein


Mass: 184506.188 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q9DDA7
#13: 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
#17: Protein Actin, cytoplasmic 1 / / Beta-actin / Cytoplasmic beta-actin


Mass: 41812.684 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: O93400

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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 gamma-Tubulin Ring Complex / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 2.2 MDa / Experimental value: NO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
11 mMmagnesium chlorideMgCl21
21 mMGuanosine triphosphateGTP1
3100 mMSodium ChlorideNaClSodium chloride1
41 mMEthylene glycol tetraacetic acidEGTA1
550 mMpiperazineethanesulfonic acidHEPES1
60.02 %Tween 20C58H114O261
70.1 mg/mLgamma-tubulin antigenic C-terminal peptide21
SpecimenConc.: 0.011 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / 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: 42000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 46 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 4 / Num. of real images: 9463

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameVersionCategory
1RELION3.0 Betaparticle selection
2SerialEM3.7image acquisition
4RELION3.0 BetaCTF correction
7Coot0.9model fitting
8UCSF Chimera1.13.1model fitting
10PHENIX1.14model refinement
11RELION3.0 Betainitial Euler assignment
12RELION3.0 Betafinal Euler assignment
13RELION3.0 Betaclassification
14RELION3.0 Beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5482328
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46096 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3RIP

3rip

RefinementStereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047122614
ELECTRON MICROSCOPYf_angle_d1.0508166080
ELECTRON MICROSCOPYf_chiral_restr0.059718665
ELECTRON MICROSCOPYf_plane_restr0.007421405
ELECTRON MICROSCOPYf_dihedral_angle_d15.132573852

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