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- EMDB-1731: The microtubule nucleating gamma-tubulin small complex assembles ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1731
TitleThe microtubule nucleating gamma-tubulin small complex assembles into a structure with microtubule-like thirteen fold symmetry
Map dataThis is the map of the gamma-tubulin small complex - Spc110p filament. The filament axis is along the Z axis.
Sample
  • Sample: Gamma-tubulin small complex in complex with Spc110p(1-220)
  • Protein or peptide: Tub4p
  • Protein or peptide: Spc97p
  • Protein or peptide: Spc98p
  • Protein or peptide: Spc110p
KeywordsMicrotubule / nucleation / tubulin / filament
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 / 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 ...: / : / Gamma tubulin / 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 SPC97 / Tubulin gamma chain / Spindle pole body component SPC98
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 8.0 Å
AuthorsKollman JM / Polka JK / Zelter A / Davis TN / Agard DA
CitationJournal: Nature / Year: 2010
Title: Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.
Authors: Justin M Kollman / Jessica K Polka / Alex Zelter / Trisha N Davis / David A Agard /
Abstract: Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory ...Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.
History
DepositionMay 14, 2010-
Header (metadata) releaseJun 28, 2010-
Map releaseNov 1, 2010-
UpdateDec 23, 2015-
Current statusDec 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-1731.jpg

Downloads & links

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Map

FileDownload / File: emd_1731.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the map of the gamma-tubulin small complex - Spc110p filament. The filament axis is along the Z axis.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 320 pix.
= 380.8 Å		1.19 Å/pix.
x 320 pix.
= 380.8 Å		1.19 Å/pix.
x 320 pix.
= 380.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.19 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-3.00519 - 6.27779
Average (Standard dev.)1.00001 (±0.999949)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 380.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.191.191.19
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z380.800380.800380.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-3.0056.2781.000

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Supplemental data

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Sample components

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Entire : Gamma-tubulin small complex in complex with Spc110p(1-220)

EntireName: Gamma-tubulin small complex in complex with Spc110p(1-220)
Components
  • Sample: Gamma-tubulin small complex in complex with Spc110p(1-220)
  • Protein or peptide: Tub4p
  • Protein or peptide: Spc97p
  • Protein or peptide: Spc98p
  • Protein or peptide: Spc110p

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Supramolecule #1000: Gamma-tubulin small complex in complex with Spc110p(1-220)

SupramoleculeName: Gamma-tubulin small complex in complex with Spc110p(1-220)
type: sample / ID: 1000
Details: The stoichiometry of gamma-TuSC to Spc110p is not known.
Number unique components: 4

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Macromolecule #1: Tub4p

MacromoleculeName: Tub4p / type: protein_or_peptide / ID: 1 / Name.synonym: gamma-tubulin / Number of copies: 2 / Oligomeric state: Helical filament / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Spindle pole body
Molecular weightTheoretical: 52.6 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (baculovirus infected) / Recombinant plasmid: pAZ37
SequenceInterPro: Gamma tubulin

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Macromolecule #2: Spc97p

MacromoleculeName: Spc97p / type: protein_or_peptide / ID: 2 / Name.synonym: Spindle pole body component p97 / Number of copies: 1 / Oligomeric state: Helical filament / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker'syeast / Location in cell: Spindle pole body
Molecular weightTheoretical: 96.8 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (baculovirus infected) / Recombinant plasmid: pDV45
SequenceInterPro: INTERPRO: IPR015698

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Macromolecule #3: Spc98p

MacromoleculeName: Spc98p / type: protein_or_peptide / ID: 3 / Name.synonym: Spindle pole body component 98p / Number of copies: 1 / Oligomeric state: Helical filament / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Spindle pole body
Molecular weightTheoretical: 98.3 MDa
Recombinant expressionOrganism: Spodoptera frugiperda (baculovirus infected) / Recombinant plasmid: pDV46
SequenceInterPro: INTERPRO: IPR015696

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Macromolecule #4: Spc110p

MacromoleculeName: Spc110p / type: protein_or_peptide / ID: 4 / Name.synonym: Spc110p
Details: Construct includes the first 220 residues of Spc110p
Number of copies: 1 / Oligomeric state: Helical filament / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Spindle pole body
Molecular weightTheoretical: 23 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (baculovirus infected) / Recombinant plasmid: pAZ34

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.6
Details: 100 mM KCl, 1mM GTP, 1mM MgCl2, 2mM EGTA, 40 mM Hepes
StainingType: NEGATIVE
Details: Sample was applied to C-FLAT holey carbon grids, blotted, and flash frozen in liquid ethane
GridDetails: 200 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blotted 3s before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Oxford side-entry cryo stage / Specimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Astigmatism corrected at 250,000 times magnification
DateSep 23, 2009
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Whole micrograph
Final angle assignmentDetails: SPIDER:theta 90 degrees, phi 90 degrees
Final reconstructionApplied symmetry - Helical parameters - Δz: 22.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 54.3 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider

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