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- EMDB-19862: Minus end of the vertebrate gamma-tubulin ring complex-capped mic... -

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Basic information

Entry
Database: EMDB / ID: EMD-19862
TitleMinus end of the vertebrate gamma-tubulin ring complex-capped microtubule
Map dataCryo-EM reconstruction of the minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
Sample
  • Complex: Minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
    • Protein or peptide: Tubulin alpha chain
    • Protein or peptide: Tubulin beta-4 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOL
Keywordscytoskeleton / microtubule / microtubule nucleation / complex / template / cap / gamma-tubulin / gamma-tubulin ring complex / CELL CYCLE
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / 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-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / 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
Tubulin beta-4 chain / Tubulin alpha chain
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 23.0 Å
AuthorsVermeulen BJA / Pfeffer S
Funding support Germany, 6 items
OrganizationGrant numberCountry
German Research Foundation (DFG)PF 963/1-4 Germany
German Research Foundation (DFG)Schi 295/4-4 Germany
Other private
German Research Foundation (DFG)INST 35/1314-1 FUGG Germany
German Research Foundation (DFG)INST 35/1503-1 FUGG Germany
German Research Foundation (DFG)INST 35/1134-1 FUGG Germany
CitationJournal: EMBO J / Year: 2024
Title: γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end.
Authors: Bram Ja Vermeulen / Anna Böhler / Qi Gao / Annett Neuner / Erik Župa / Zhenzhen Chu / Martin Würtz / Ursula Jäkle / Oliver J Gruss / Stefan Pfeffer / Elmar Schiebel /
Abstract: The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure ...The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.
History
DepositionMar 15, 2024-
Header (metadata) releaseNov 6, 2024-
Map releaseNov 6, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19862.png

Downloads & links

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Map

FileDownload / File: emd_19862.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 256 pix.
= 513.024 Å		2 Å/pix.
x 256 pix.
= 513.024 Å		2 Å/pix.
x 256 pix.
= 513.024 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.004 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0157
Minimum - Maximum-0.020712815 - 0.050880447
Average (Standard dev.)-0.00009258807 (±0.005599869)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 513.024 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map for the cryo-EM reconstruction of the...

Fileemd_19862_half_map_1.map
AnnotationHalf map for the cryo-EM reconstruction of the minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map for the cryo-EM reconstruction of the...

Fileemd_19862_half_map_2.map
AnnotationHalf map for the cryo-EM reconstruction of the minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Minus end of the vertebrate gamma-tubulin ring complex-capped mic...

EntireName: Minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
Components
  • Complex: Minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
    • Protein or peptide: Tubulin alpha chain
    • Protein or peptide: Tubulin beta-4 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOL

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Supramolecule #1: Minus end of the vertebrate gamma-tubulin ring complex-capped mic...

SupramoleculeName: Minus end of the vertebrate gamma-tubulin ring complex-capped microtubule
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Minus end of the vertebrate gamma-tubulin ring complex-capped microtubule induced through the RanGTP spindle assembly pathway and isolated from Xenopus laevis egg extract
Source (natural)Organism: Xenopus laevis (African clawed frog)

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Macromolecule #1: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 23 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 50.120297 KDa
SequenceString: MRECISVHVG QAGVQIGNSC WELYCLEHGL QPDGTMPSEK SATMVDSSFG TFFSETGSGK HVPRAVFVDL EQTVIGEIRN GPYRSLFHP EQLITGKEDA ANNYARGHYT IGKELIDSVL DRVRKMADQC SGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISVHVG QAGVQIGNSC WELYCLEHGL QPDGTMPSEK SATMVDSSFG TFFSETGSGK HVPRAVFVDL EQTVIGEIRN GPYRSLFHP EQLITGKEDA ANNYARGHYT IGKELIDSVL DRVRKMADQC SGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSVYPAPRIS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICNRNLD IERPSYTNLN RLIAQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLVTYSPIIS AEKAYHEQLS VPEITNACFE YSNQMVKCDP RRGKYMACCL LYR GDVVPK DVNAAIAAIK TRRSIQFVDW CPTGFKVGIN YQPPTAVPGG DVAKVLRAVC MLSNTTAIAE AWARLDHKFD LMYS KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GTESGDGGED EEDEY

UniProtKB: Tubulin alpha chain

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Macromolecule #2: Tubulin beta-4 chain

MacromoleculeName: Tubulin beta-4 chain / type: protein_or_peptide / ID: 2 / Number of copies: 19 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 49.86277 KDa
SequenceString: MREIVHLQAG QCGNQIGAKF WEVISDEHGI DPTGAYHGDS DLQLERINVY YNEATGGKYV PRAVLVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKEAESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHLQAG QCGNQIGAKF WEVISDEHGI DPTGAYHGDS DLQLERINVY YNEATGGKYV PRAVLVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKEAESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAIF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATAEEEGE FEEGEEEENA

UniProtKB: Tubulin beta-4 chain

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Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 23 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 23 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 19 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Macromolecule #6: TAXOL

MacromoleculeName: TAXOL / type: ligand / ID: 6 / Number of copies: 19 / Formula: TA1
Molecular weightTheoretical: 853.906 Da
Chemical component information

ChemComp-TA1:
TAXOL / medication, chemotherapy*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 33000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

10491


Details: GRIP2 and gamma-tubulin in spokes 5 and 6 were removed
Final reconstructionResolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 8497
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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