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- EMDB-5989: Electron cryo-tomography of the Microtubule Nucleation Complex in... -

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Basic information

Entry
Database: EMDB / ID: EMD-5989
TitleElectron cryo-tomography of the Microtubule Nucleation Complex in the Yeast Spindle Pole Body
Map dataSubtomogram average of the microtubule minus ends attached the purified yeast spindle pole body
Sample
  • Sample: yeast spindle pole body
  • Organelle or cellular component: Spindle Pole Body
Keywordsmicrotubule / nucleation / gamma-tubulin / yeast
Biological speciesSaccharomyces uvarum (yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 38.0 Å
AuthorsKollman JM / Greenberg C / Li S / Fernandez JJ / Moritz M / Zelter A / Fong K / Sali A / Kilmartin JV / 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
DepositionJun 13, 2014-
Header (metadata) releaseJul 30, 2014-
Map releaseJan 21, 2015-
UpdateJan 21, 2015-
Current statusJan 21, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5989.gif

Downloads & links

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Map

FileDownload / File: emd_5989.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of the microtubule minus ends attached the purified yeast spindle pole body
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.64 Å/pix.
x 80 pix.
= 851.2 Å		10.64 Å/pix.
x 80 pix.
= 851.2 Å		10.64 Å/pix.
x 80 pix.
= 851.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 10.64 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.5 / Movie #1: 6.5
Minimum - Maximum-94.773666379999995 - 105.956817630000003
Average (Standard dev.)0.0 (±5.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40120
Dimensions808080
Spacing808080
CellA=B=C: 851.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z10.6410.6410.64
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z851.200851.200851.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-40-40120
NC/NR/NS808080
D min/max/mean-94.774105.957-0.000

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Supplemental data

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Sample components

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Entire : yeast spindle pole body

EntireName: yeast spindle pole body
Components
  • Sample: yeast spindle pole body
  • Organelle or cellular component: Spindle Pole Body

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Supramolecule #1000: yeast spindle pole body

SupramoleculeName: yeast spindle pole body / type: sample / ID: 1000
Oligomeric state: 7 gamma-TuSC complexes form a one-turn spiral
Number unique components: 1

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Supramolecule #1: Spindle Pole Body

SupramoleculeName: Spindle Pole Body / type: organelle_or_cellular_component / ID: 1 / Name.synonym: microtubule organizing center / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces uvarum (yeast) / synonym: budding yeast / Organelle: spindle pole body / Location in cell: nuclear membrane

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 6.5 / Details: 10 mM Bis-Tris-Cl, 0.1 mM MgCl2, 20% v/v DMSO
GridDetails: 300 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
DateMay 14, 2010
Image recordingCategory: CCD / Film or detector model: OTHER / Digitization - Sampling interval: 1.5 µm / Number real images: 82 / Average electron dose: 60 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 56391 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 41000
Sample stageSpecimen holder: Nitrogen-cooled / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe subtomograms were selected manually.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 38.0 Å / Resolution method: OTHER / Software - Name: Xmipp / Number subtomograms used: 1156
CTF correctionDetails: each image, Wiener filter

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