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- PDB-7m2w: Engineered disulfide cross-linked closed conformation of the Yeas... -

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Basic information

Entry
Database: PDB / ID: 7m2w
TitleEngineered disulfide cross-linked closed conformation of the Yeast gamma-TuRC(SS)
Components
  • Spindle pole body component 110
  • Spindle pole body component SPC97
  • Spindle pole body component SPC98
  • Tubulin gamma chain
KeywordsCELL CYCLE / microtubule nucleation
Function / homology
Function and homology information


gamma-tubulin complex localization to nuclear side of mitotic spindle pole body / protein localization to mitotic spindle pole body / microtubule nucleation by spindle pole body / gamma-tubulin small complex => GO:0008275 / inner plaque of spindle pole body / outer plaque of spindle pole body / central plaque of spindle pole body / gamma-tubulin small complex / equatorial microtubule organizing center / karyogamy involved in conjugation with cellular fusion ...gamma-tubulin complex localization to nuclear side of mitotic spindle pole body / protein localization to mitotic spindle pole body / microtubule nucleation by spindle pole body / gamma-tubulin small complex => GO:0008275 / inner plaque of spindle pole body / outer plaque of spindle pole body / central plaque of spindle pole body / gamma-tubulin small complex / equatorial microtubule organizing center / karyogamy involved in conjugation with cellular fusion / mitotic spindle pole body / mitotic spindle elongation / gamma-tubulin complex / meiotic spindle organization / positive regulation of cytoplasmic translation / microtubule nucleation / positive regulation of microtubule nucleation / gamma-tubulin binding / spindle pole body / mitotic sister chromatid segregation / cytoplasmic microtubule organization / spindle assembly / meiotic cell cycle / microtubule cytoskeleton organization / structural constituent of cytoskeleton / mitotic spindle organization / spindle / spindle pole / mitotic cell cycle / protein-containing complex assembly / microtubule / calmodulin binding / GTPase activity / GTP binding / protein-containing complex binding / nucleus / cytoplasm
Similarity search - Function
Spindle pole body component 110, C-terminal / Spindle pole body component 110 C-terminal domain / Gamma tubulin / Gamma-tubulin complex, C-terminal domain superfamily / Gamma-tubulin complex component protein / Gamma tubulin complex component, C-terminal / Gamma tubulin complex component protein, N-terminal / Gamma tubulin complex component C-terminal / Gamma tubulin complex component N-terminal / Tubulin, C-terminal ...Spindle pole body component 110, C-terminal / Spindle pole body component 110 C-terminal domain / Gamma tubulin / Gamma-tubulin complex, C-terminal domain superfamily / Gamma-tubulin complex component protein / Gamma tubulin complex component, C-terminal / Gamma tubulin complex component protein, N-terminal / Gamma tubulin complex component C-terminal / Gamma tubulin complex component N-terminal / Tubulin, C-terminal / Tubulin / Tubulin C-terminal domain / Tubulin subunits alpha, beta, and gamma signature. / Tubulin, conserved site / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal / 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 chain / GUANOSINE-5'-TRIPHOSPHATE / Spindle pole body component SPC97 / Spindle pole body component 110 / Spindle pole body component SPC98
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3 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

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Assembly

Deposited unit
B: Tubulin gamma chain
A: Tubulin gamma chain
C: Tubulin gamma chain
D: Tubulin gamma chain
E: Spindle pole body component SPC97
F: Spindle pole body component SPC98
G: Spindle pole body component SPC97
H: Spindle pole body component SPC98
U: Spindle pole body component 110
K: Spindle pole body component 110
X: Spindle pole body component 110
Y: Spindle pole body component 110
hetero molecules


Theoretical massNumber of molelcules
Total (without water)705,37816
Polymers703,28512
Non-polymers2,0934
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area61890 Å2
ΔGint-264 kcal/mol
Surface area191590 Å2

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Components

#1: Protein
Tubulin gamma chain / Gamma-tubulin


Mass: 52733.340 Da / Num. of mol.: 4 / Mutation: S58C, G288C
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 SPC97 /


Mass: 96940.594 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: SPC97, YHR172W / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P38863
#3: Protein Spindle pole body component SPC98 /


Mass: 98336.211 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: SPC98, YNL126W, N1222, N1879 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P53540
#4: Protein
Spindle pole body component 110 / / Extragenic suppressor of CMD1-1 mutant protein 1 / Nuclear filament-related protein 1 / Spindle ...Extragenic suppressor of CMD1-1 mutant protein 1 / Nuclear filament-related protein 1 / Spindle pole body spacer protein SPC110


Mass: 25449.555 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SPC110, NUF1, XCM1, YDR356W, D9476.3 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P32380
#5: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, 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: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Engineered disulfide cross-linked closed conformation of the Yeast gamma-TuRC(SS)
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast) / Cellular location: Spindle Pole Body / Organelle: Nucleus
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 mMOxidized Glutathione1
60.5 mMGTP1
72.5 %v/vGlycerol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 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 / Calibrated magnification: 47214 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 72 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3024
Image scansMovie frames/image: 120

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN2particle selectione2helixboxer.py
2SerialEMimage acquisition
4cisTEM1.0 betaCTF correction
7PHENIXmodel fitting
8Cootmodel fitting
9Rosettamodel fitting
11RELIONinitial Euler assignment
12cisTEM1.0 Betafinal Euler assignment
13cisTEM1.0 BetaclassificationFocused classification
14cisTEM1.0 Beta3D reconstruction
15PHENIXmodel refinement
16Cootmodel refinement
17Rosettamodel refinement
CTF correctionDetails: Final reconstruction in cisTEM / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 0 ° / Axial rise/subunit: 0.1 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 175500
Details: Boxed approximately every 3 asymmetric units using a rise of 21 Angstroms, with a box size of 600 pixels (635.4A).
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 148911 / Algorithm: FOURIER SPACE
Details: Final reconstruction is on symmetry expanded particles.
Num. of class averages: 4 / Symmetry type: HELICAL
Atomic model buildingB value: 60 / Protocol: OTHER / Space: REAL

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