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- PDB-7m2z: Monomeric single-particle reconstruction of the Yeast gamma-TuSC -

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Basic information

Entry
Database: PDB / ID: 7m2z
TitleMonomeric single-particle reconstruction of the Yeast gamma-TuSC
Components
  • Spindle pole body component SPC97
  • Spindle pole body component SPC98
  • Tubulin gamma chain
KeywordsCELL CYCLE / microtubule nucleation
Function / homology
Function and homology information


inner plaque of spindle pole body / microtubule nucleation by spindle pole body / outer plaque of spindle pole body / gamma-tubulin small complex / regulation of microtubule nucleation / mitotic spindle pole body / equatorial microtubule organizing center / gamma-tubulin complex / meiotic spindle organization / microtubule nucleation ...inner plaque of spindle pole body / microtubule nucleation by spindle pole body / outer plaque of spindle pole body / gamma-tubulin small complex / regulation of microtubule nucleation / mitotic spindle pole body / equatorial microtubule organizing center / gamma-tubulin complex / meiotic spindle organization / microtubule nucleation / positive regulation of cytoplasmic translation / spindle pole body / gamma-tubulin binding / mitotic sister chromatid segregation / spindle assembly / cytoplasmic microtubule organization / mitotic spindle organization / meiotic cell cycle / structural constituent of cytoskeleton / spindle / spindle pole / mitotic cell cycle / microtubule / GTP binding / nucleus / cytoplasm
Similarity search - Function
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 ...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
GUANOSINE-5'-DIPHOSPHATE / Spindle pole body component SPC97 / Tubulin gamma chain / Spindle pole body component SPC98
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBrilot, A.F. / Lyon, A.S. / Zelter, A. / Viswanath, S. / Maxwell, A. / MacCoss, M.J. / Muller, E.G. / Sali, A. / Davis, T.N. / Agard, D.A.
Funding support United States, 13items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)David Agard United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM031627 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118099 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01 GM105537 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103533 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM083960 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM109824 United States
National Science Foundation (NSF, United States)1144247 United States
National Institutes of Health/Office of the Director1S10OD020054 United States
National Institutes of Health/Office of the Director1S10OD021741 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM124149 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30 GM124169 United States
Department of Energy (DOE, United States)DE-AC02-05CH11231 United States
CitationJournal: Elife / Year: 2021
Title: CM1-driven assembly and activation of yeast γ-tubulin small complex underlies microtubule nucleation.
Authors: Axel F Brilot / Andrew S Lyon / Alex Zelter / Shruthi Viswanath / Alison Maxwell / Michael J MacCoss / Eric G Muller / Andrej Sali / Trisha N Davis / David A Agard /
Abstract: Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In , γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub- ...Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In , γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies, which associate helically to template MT growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC.
History
DepositionMar 17, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 12, 2021Provider: repository / Type: Initial release
Revision 1.1May 19, 2021Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Movie
  • Deposited structure unit
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  • EMDB-23638
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Assembly

Deposited unit
B: Tubulin gamma chain
A: Tubulin gamma chain
C: Spindle pole body component SPC98
D: Spindle pole body component SPC97
hetero molecules


Theoretical massNumber of molelcules
Total (without water)301,5066
Polymers300,6194
Non-polymers8862
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, mass spectrometry, microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18520 Å2
ΔGint-86 kcal/mol
Surface area105780 Å2

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Components

#1: Protein Tubulin gamma chain / Gamma-tubulin


Mass: 52671.188 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: TUB4, YLR212C, L8167.21 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P53378
#2: Protein Spindle pole body component SPC98 /


Mass: 98336.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SPC98, YNL126W, N1222, N1879 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P53540
#3: Protein Spindle pole body component SPC97 /


Mass: 96940.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SPC97, YHR172W / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P38863
#4: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
Has ligand of interestN

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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: Monomer of the Yeast gamma-TuSC / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Cellular location: Spindle Pole Body
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: SF9
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
140 mMHEPES-KOH1
2100 mMPotassium Chloride1
32 mMMagnesium Chloride1
41 mMEGTA1
51 mMGDP1
62.5 %v/vGlycerol1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was a mixture of monomers and dimers.
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Whatman #1 Filter papers used.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Calibrated magnification: 39891 X / Nominal defocus max: 1000 nm / Nominal defocus min: 400 nm / Calibrated defocus min: 3000 nm / Calibrated defocus max: 5000 nm / Cs: 2 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 20 sec. / Electron dose: 76 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansSampling size: 2.5 µm / Movie frames/image: 100 / Used frames/image: 3-100

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Processing

EM software
IDNameVersionCategory
1cisTEM1.0 betaparticle selection
2SerialEMimage acquisition
3Leginonimage acquisition
5cisTEM1.0 betaCTF correction
8PHENIXmodel fitting
9Cootmodel fitting
10Rosettamodel fitting
12cisTEM1.0 betainitial Euler assignment
13cisTEM1.0 Betafinal Euler assignment
14cisTEM1.0 Betaclassification
15cisTEM1.0 Beta3D reconstruction
16PHENIXmodel refinement
17Cootmodel refinement
18Rosettamodel refinement
CTF correctionDetails: Final reconstruction in cisTEM / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3210917 / Details: cisTEM picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 398841 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 164 / Protocol: OTHER / Space: REAL

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