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- EMDB-23635: Engineered disulfide cross-linked closed conformation of the Yeas... -

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Basic information

Entry
Database: EMDB / ID: EMD-23635
TitleEngineered disulfide cross-linked closed conformation of the Yeast gamma-TuRC(SS)
Map data
SampleEngineered disulfide cross-linked closed conformation of the Yeast gamma-TuRC(SS):
Tubulin gamma chain / (Spindle pole body component ...) x 3 / ligand
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 / gamma-tubulin small complex => GO:0008275 / 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 / 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 / gamma-tubulin small complex => GO:0008275 / 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 / 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, N-terminal / Gamma tubulin complex component, C-terminal / Gamma-tubulin complex component protein / Gamma tubulin complex component C-terminal / Gamma tubulin complex component N-terminal / Tubulin C-terminal domain ...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, N-terminal / Gamma tubulin complex component, C-terminal / Gamma-tubulin complex component protein / Gamma tubulin complex component C-terminal / Gamma tubulin complex component N-terminal / Tubulin C-terminal domain / Tubulin, C-terminal / Tubulin / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ family, 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 (baker's yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodhelical reconstruction / cryo EM / Resolution: 3 Å
AuthorsBrilot AF / Lyon AS / Zelter A / Viswanath S / Maxwell A / MacCoss MJ / Muller EG / Sali A / Davis TN / Agard DA
Funding support United States, 13 items
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, 2021-
Header (metadata) releaseMay 12, 2021-
Map releaseMay 12, 2021-
UpdateMay 19, 2021-
Current statusMay 19, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7m2w
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7m2w
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_23635.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 600 pix.
= 635.4 Å
1.06 Å/pix.
x 600 pix.
= 635.4 Å
1.06 Å/pix.
x 600 pix.
= 635.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum-18.01994 - 32.981194
Average (Standard dev.)0.003294716 (±0.6316574)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 635.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z635.400635.400635.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-18.02032.9810.003

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Supplemental data

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Half map: gamma-TuRC(SS) half map 2

Fileemd_23635_half_map_1.map
Annotationgamma-TuRC(SS) half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: gamma-TuRC(SS) half map 1

Fileemd_23635_half_map_2.map
Annotationgamma-TuRC(SS) half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Engineered disulfide cross-linked closed conformation of the Yeas...

EntireName: Engineered disulfide cross-linked closed conformation of the Yeast gamma-TuRC(SS)
Number of components: 6

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Component #1: protein, Engineered disulfide cross-linked closed conformation of...

ProteinName: Engineered disulfide cross-linked closed conformation of the Yeast gamma-TuRC(SS)
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Strain: SF9
Source (natural)Organelle: Nucleus / Location in cell: Spindle Pole Body

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Component #2: protein, Tubulin gamma chain

ProteinName: Tubulin gamma chain / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 52.73334 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, Spindle pole body component SPC97

ProteinName: Spindle pole body component SPC97 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 96.940594 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #4: protein, Spindle pole body component SPC98

ProteinName: Spindle pole body component SPC98 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 98.336211 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #5: protein, Spindle pole body component 110

ProteinName: Spindle pole body component 110 / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 25.449555 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #6: ligand, GUANOSINE-5'-TRIPHOSPHATE

LigandName: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.52318 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 0.1 Å / Delta phi: 0 %deg;
Sample solutionpH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 100 % / Details: Whatman #1 Filter papers used..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 72 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 47214.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 600.0 - 2000.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3024

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: cisTEM / CTF correction: Final reconstruction in cisTEM / Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Final reconstruction is on symmetry expanded particles.
Euler angles: Projection matching in cisTEM

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Atomic model buiding

Modeling #1Refinement space: REAL / Overall bvalue: 60
Output model

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