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- EMDB-2799: Cryo-EM structure of gamma-TuSC oligomers in a closed conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-2799
TitleCryo-EM structure of gamma-TuSC oligomers in a closed conformation
Map dataReconstruction of yeast gamma-TuSC trapped in a closed state by disulfide crosslinks
Sample
  • Sample: Recombinant yeast gamma-TuSC mutant S58C/G288C
  • Protein or peptide: gamma tubulin S58C/G288C
  • Protein or peptide: GCP2GOLGA4
  • Protein or peptide: GCP3
  • Protein or peptide: Spc110 (1-220)
KeywordsMicrotubule nucleation / gamma tubulin
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 pole body ...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 pole body / equatorial microtubule organizing center / mitotic spindle elongation / gamma-tubulin complex / meiotic spindle organization / positive regulation of microtubule nucleation / 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 / protein-containing complex assembly / microtubule / calmodulin binding / protein-containing complex binding / GTP 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 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 ...Spindle pole body component 110, C-terminal / Spindle pole body component 110 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
Spindle pole body component 110 / Spindle pole body component SPC97 / Tubulin gamma chain / Spindle pole body component SPC98
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodhelical reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsKollman JM / Greenberg CH / Li S / Moritz M / Zelter A / Fong K / Fernandez J-J / Sali A / Kilmartin J / Davis TN / Agard DA
CitationJournal: Nat Struct Mol Biol / Year: 2015
Title: Ring closure activates yeast γTuRC for species-specific microtubule nucleation.
Authors: Justin M Kollman / Charles H Greenberg / Sam Li / Michelle Moritz / Alex Zelter / Kimberly K Fong / Jose-Jesus Fernandez / Andrej Sali / John Kilmartin / Trisha N Davis / David A Agard /
Abstract: The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-tubulin small complex (γTuSC) subunits and is thought to function as a ...The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm the functional importance of the closed γTuSC ring, we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, thus suggesting that this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a strong preference for tubulin from the same species.
History
DepositionOct 15, 2014-
Header (metadata) releaseOct 22, 2014-
Map releaseJan 21, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

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  • Atomic models: PDB-5flz
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  • Surface view with fitted model
  • Atomic models: PDB-5flz
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5flz
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Structure viewerEM map:
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Supplemental images

emd-2799_map...

Downloads & links

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Map

FileDownload / File: emd_2799.map.gz / Format: CCP4 / Size: 35.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of yeast gamma-TuSC trapped in a closed state by disulfide crosslinks
Voxel sizeX=Y=Z: 1.88 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.94397974 - 3.0450983
Average (Standard dev.)0.0 (±0.31405976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-106-106-106
Dimensions212212212
Spacing212212212
CellA=B=C: 398.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.881.881.88
M x/y/z212212212
origin x/y/z0.0000.0000.000
length x/y/z398.560398.560398.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-106-106-106
NC/NR/NS212212212
D min/max/mean-1.9443.0450.000

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Supplemental data

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Sample components

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Entire : Recombinant yeast gamma-TuSC mutant S58C/G288C

EntireName: Recombinant yeast gamma-TuSC mutant S58C/G288C
Components
  • Sample: Recombinant yeast gamma-TuSC mutant S58C/G288C
  • Protein or peptide: gamma tubulin S58C/G288C
  • Protein or peptide: GCP2GOLGA4
  • Protein or peptide: GCP3
  • Protein or peptide: Spc110 (1-220)

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Supramolecule #1000: Recombinant yeast gamma-TuSC mutant S58C/G288C

SupramoleculeName: Recombinant yeast gamma-TuSC mutant S58C/G288C / type: sample / ID: 1000 / Oligomeric state: heteropentamer / Number unique components: 4

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Macromolecule #1: gamma tubulin S58C/G288C

MacromoleculeName: gamma tubulin S58C/G288C / type: protein_or_peptide / ID: 1 / Name.synonym: tub4
Details: Cysteine residues were introduced at positions 58 and 288 to promote crosslinking of the helical complex.
Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: spindle pole body
Molecular weightTheoretical: 55 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceUniProtKB: Tubulin gamma chain

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Macromolecule #2: GCP2

MacromoleculeName: GCP2 / type: protein_or_peptide / ID: 2 / Name.synonym: Spc97 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: spindle pole body
Molecular weightTheoretical: 97 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceUniProtKB: Spindle pole body component SPC97

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Macromolecule #3: GCP3

MacromoleculeName: GCP3 / type: protein_or_peptide / ID: 3 / Name.synonym: Spc98 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: spindle pole body
Molecular weightTheoretical: 98 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceUniProtKB: Spindle pole body component SPC98

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Macromolecule #4: Spc110 (1-220)

MacromoleculeName: Spc110 (1-220) / type: protein_or_peptide / ID: 4
Details: Residues 1-220 of Spc110 were expressed with an N-terminal GST tagged, which was cleaved off during purification.
Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: spindle pole body
Molecular weightTheoretical: 25 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceUniProtKB: Spindle pole body component 110

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.6
Details: 40 mM Hepes PH 7.6, 100 mM KCl, 1 mM EGTA, 1mM MgCl2, 1 mM oxidized glutathione
GridDetails: 400 mesh C-FLAT 2/2 grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK I / Method: Blot for 2-5 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 94000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.12 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 135,000 time magnifiaction
DateMay 25, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 364 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each micrograph
Final reconstructionApplied symmetry - Helical parameters - Δz: 22.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 54.3 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: OTHER / Software - Name: SPIDER, hsearch_lorentz, EMAN1, ctffind
DetailsIHRSR was carried out in SPIDER, using hsearch_lorentz to search for helical symmetry parameters in unsymmetrized maps.

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