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- PDB-8rx1: CryoEM structure of the gTuRC-CM1dim complex -

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Basic information

Entry
Database: PDB / ID: 8rx1
TitleCryoEM structure of the gTuRC-CM1dim complex
Components
  • (Gamma-tubulin complex component ...) x 5
  • (Mitotic-spindle organizing protein ...) x 2
  • Actin b
  • CM1
  • Tubulin gamma-1 chain
KeywordsSTRUCTURAL PROTEIN / gTuRC / gTuSC / CM1 / CDK5RAP2 / gamma-tubulin / microtubule / alfa/beta-tubulin nucleation / cytoskeleton
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
MOZART2 family / Mitotic-spindle organizing gamma-tubulin ring associated / 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 ...MOZART2 family / Mitotic-spindle organizing gamma-tubulin ring associated / 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 / Mitotic-spindle organizing protein 2A / 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)
Spodoptera frugiperda (fall armyworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsLlorca, O. / Serna, M. / Gonzalez-Rodriguez, N.
Funding support Spain, European Union, 2items
OrganizationGrant numberCountry
Agencia Estatal de Investigacion (AEI)AEI/10.13039/501100011 003 Spain
European Regional Development FundPID2020-114429RB-I00European Union
CitationJournal: Dev Cell / Year: 2024
Title: CDK5RAP2 activates microtubule nucleator γTuRC by facilitating template formation and actin release.
Authors: Marina Serna / Fabian Zimmermann / Chithran Vineethakumari / Nayim Gonzalez-Rodriguez / Oscar Llorca / Jens Lüders /
Abstract: To organize microtubules, cells tightly control the activity of the microtubule nucleator γ-tubulin ring complex (γTuRC). The open ring-shaped γTuRC was proposed to nucleate microtubules by a ...To organize microtubules, cells tightly control the activity of the microtubule nucleator γ-tubulin ring complex (γTuRC). The open ring-shaped γTuRC was proposed to nucleate microtubules by a template mechanism. However, its splayed structure does not match microtubule symmetry, leaving it unclear how γTuRC becomes an efficient nucleator. Here, we identify the mechanism of γTuRC activation by CDK5RAP2 centrosomin motif 1 (CM1). Using cryoelectron microscopy (cryo-EM), we find that activation involves binding of multiple CM1 dimers to five distinct sites around the outside of the γTuRC cone, which crucially depends on regulatory modules formed by MZT2 and the N-terminal extensions of GCP2 subunits. CM1 binding promotes lateral interactions between GCP subunits to facilitate microtubule-like conformations and release of luminal actin that is integral to non-activated γTuRC. We propose a model where generation of γTuRC with an expanded conformational range, rather than perfect symmetry, is sufficient to boost nucleation activity.
History
DepositionFeb 6, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 2, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Oct 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

Downloads & links

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Assembly

Deposited unit
1: Tubulin gamma-1 chain
2: Tubulin gamma-1 chain
7: Actin b
A: Gamma-tubulin complex component 2
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: CM1
H: CM1
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
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
a: Mitotic-spindle organizing protein 1
b: Gamma-tubulin complex component 3
c: Mitotic-spindle organizing protein 1
d: Gamma-tubulin complex component 6
e: Mitotic-spindle organizing protein 2A
f: Gamma-tubulin complex component 2
g: CM1
h: CM1
i: CM1
j: CM1
k: Gamma-tubulin complex component 2
n: Gamma-tubulin complex component 3
s: CM1
t: CM1
u: CM1
v: CM1


Theoretical massNumber of molelcules
Total (without water)2,969,31545
Polymers2,969,31545
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

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Protein , 3 types, 25 molecules 12OPQRSTUVWXYZ7GHghijstuv

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


Mass: 51227.770 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBG1, TUBG / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P23258
#2: Protein Actin b


Mass: 40697.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Spodoptera frugiperda (fall armyworm)
#5: Protein
CM1


Mass: 26765.963 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)

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Gamma-tubulin complex component ... , 5 types, 17 molecules ACEMfkBDFNbnIKJLd

#3: 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.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBGCP2, GCP2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BSJ2
#4: 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 frugiperda (fall armyworm) / References: UniProt: Q96CW5
#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 frugiperda (fall armyworm) / References: UniProt: Q9UGJ1
#7: Protein Gamma-tubulin complex component 5 / GCP-5


Mass: 118467.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBGCP5, GCP5, KIAA1899 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q96RT8
#8: 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 frugiperda (fall armyworm) / References: UniProt: Q96RT7

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Mitotic-spindle organizing protein ... , 2 types, 3 molecules ace

#9: 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.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MZT1, C13orf37, MOZART1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q08AG7
#10: Protein Mitotic-spindle organizing protein 2A / Mitotic-spindle organizing protein associated with a ring of gamma-tubulin 2A


Mass: 16240.576 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MZT2A, FAM128A, MOZART2A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6P582

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Details

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: gTuRC-CM1dim complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 90 % / Chamber temperature: 277 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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 900 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 55.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 94094
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1Topaz0.2particle selection
2RELION4particle selection
3EPUimage acquisition
5CTFFIND4CTF correction
8UCSF Chimeramodel fitting
9UCSF ChimeraXmodel fitting
11cryoSPARC4.0+initial Euler assignment
12RELION4initial Euler assignment
13RELION4final Euler assignment
14RELION4classification
15RELION43D reconstruction
16REFMAC5model refinement
17PHENIX1.20.1-4487model refinement
18ISOLDE1.7model refinement
19Coot0.9.8.5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4399521
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 579078 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17AS4

7as4

17AS41PDBexperimental model
26X0V

6x0v

16X0V2PDBexperimental model

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