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Yorodumi- PDB-7m2w: Engineered disulfide cross-linked closed conformation of the Yeas... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7m2w | ||||||||||||||||||||||||||||||||||||||||||
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Title | Engineered disulfide cross-linked closed conformation of the Yeast gamma-TuRC(SS) | ||||||||||||||||||||||||||||||||||||||||||
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Keywords | CELL 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 / inner plaque of spindle pole body / microtubule nucleation by spindle pole body / outer plaque of spindle pole body / central plaque of spindle pole body / gamma-tubulin small complex / regulation of microtubule nucleation / karyogamy involved in conjugation with cellular fusion / mitotic spindle elongation ...gamma-tubulin complex localization to nuclear side of mitotic spindle pole body / protein localization to mitotic spindle pole body / inner plaque of spindle pole body / microtubule nucleation by spindle pole body / outer plaque of spindle pole body / central plaque of spindle pole body / gamma-tubulin small complex / regulation of microtubule nucleation / karyogamy involved in conjugation with cellular fusion / mitotic spindle elongation / equatorial microtubule organizing center / mitotic spindle pole body / positive regulation of microtubule nucleation / meiotic spindle organization / gamma-tubulin complex / microtubule nucleation / positive regulation of cytoplasmic translation / gamma-tubulin binding / spindle pole body / 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 / protein-containing complex assembly / microtubule / cytoskeleton / calmodulin binding / GTP binding / protein-containing complex binding / nucleus / cytoplasm Similarity search - Function | ||||||||||||||||||||||||||||||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||||||||||||||||||||||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3 Å | ||||||||||||||||||||||||||||||||||||||||||
Authors | Brilot, 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
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7m2w.cif.gz | 861.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7m2w.ent.gz | 722.8 KB | Display | PDB format |
PDBx/mmJSON format | 7m2w.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7m2w_validation.pdf.gz | 721.5 KB | Display | wwPDB validaton report |
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Full document | 7m2w_full_validation.pdf.gz | 801.8 KB | Display | |
Data in XML | 7m2w_validation.xml.gz | 109.2 KB | Display | |
Data in CIF | 7m2w_validation.cif.gz | 178.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/m2/7m2w ftp://data.pdbj.org/pub/pdb/validation_reports/m2/7m2w | HTTPS FTP |
-Related structure data
Related structure data | 23635MC 7m2xC 7m2yC 7m2zC 7m3pC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 | 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 | 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 | 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 / Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: Engineered disulfide cross-linked closed conformation of the Yeast gamma-TuRC(SS) Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||||
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Molecular weight | Units: MEGADALTONS / Experimental value: YES | ||||||||||||||||||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Cellular location: Spindle Pole Body / Organelle: Nucleus | ||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) / Strain: SF9 | ||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Whatman #1 Filter papers used. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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 scans | Movie frames/image: 120 |
-Processing
EM software |
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CTF correction | Details: Final reconstruction in cisTEM / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: 0 ° / Axial rise/subunit: 0.1 Å / Axial symmetry: C1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. 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 reconstruction | Resolution: 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 building | B value: 60 / Protocol: OTHER / Space: REAL |