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- EMDB-18193: Cross-linked human gTuSC oligomeric ring -

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Basic information

Entry
Database: EMDB / ID: EMD-18193
TitleCross-linked human gTuSC oligomeric ring
Map dataCross-linked human gTuSC
Sample
  • Complex: Cross-linked human gTuRC oligomeric ring
    • Protein or peptide: GCP2
    • Protein or peptide: GCP3
    • Protein or peptide: gamma-tubulin
KeywordsgTuSC / gTuRC / Microtubule / gamma-tubulin / alpha/beta-tubulin nucleation / STRUCTURAL PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.78 Å
AuthorsLlorca O / Serna M
Funding support Spain, European Union, 2 items
OrganizationGrant numberCountry
Agencia Estatal de Investigacion (AEI)AEI/10.13039/501100011 003 Spain
European Regional Development FundPID2020-114429RB-I00European Union
CitationJournal: Dev Cell / Year: 2024
Title: CDK5RAP2 activates microtubule nucleator γTuRC by facilitating template formation and actin release.
Authors: Marina Serna / Fabian Zimmermann / Chithran Vineethakumari / Nayim Gonzalez-Rodriguez / Oscar Llorca / Jens Lüders /
Abstract: To organize microtubules, cells tightly control the activity of the microtubule nucleator γ-tubulin ring complex (γTuRC). The open ring-shaped γTuRC was proposed to nucleate microtubules by a ...To organize microtubules, cells tightly control the activity of the microtubule nucleator γ-tubulin ring complex (γTuRC). The open ring-shaped γTuRC was proposed to nucleate microtubules by a template mechanism. However, its splayed structure does not match microtubule symmetry, leaving it unclear how γTuRC becomes an efficient nucleator. Here, we identify the mechanism of γTuRC activation by CDK5RAP2 centrosomin motif 1 (CM1). Using cryoelectron microscopy (cryo-EM), we find that activation involves binding of multiple CM1 dimers to five distinct sites around the outside of the γTuRC cone, which crucially depends on regulatory modules formed by MZT2 and the N-terminal extensions of GCP2 subunits. CM1 binding promotes lateral interactions between GCP subunits to facilitate microtubule-like conformations and release of luminal actin that is integral to non-activated γTuRC. We propose a model where generation of γTuRC with an expanded conformational range, rather than perfect symmetry, is sufficient to boost nucleation activity.
History
DepositionAug 14, 2023-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18193.png

Downloads & links

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Map

FileDownload / File: emd_18193.map.gz / Format: CCP4 / Size: 202.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCross-linked human gTuSC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.64 Å/pix.
x 376 pix.
= 614.76 Å		1.64 Å/pix.
x 376 pix.
= 614.76 Å		1.64 Å/pix.
x 376 pix.
= 614.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.635 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.012086627 - 0.043693185
Average (Standard dev.)0.0002997189 (±0.0022246873)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions376376376
Spacing376376376
CellA=B=C: 614.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18193_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map of the cross-linked human gTuSC

Fileemd_18193_additional_1.map
AnnotationSharpened map of the cross-linked human gTuSC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of the cross-linked human gTuSC

Fileemd_18193_half_map_1.map
AnnotationHalf map 1 of the cross-linked human gTuSC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of the cross-linked human gTuSC

Fileemd_18193_half_map_2.map
AnnotationHalf map 2 of the cross-linked human gTuSC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cross-linked human gTuRC oligomeric ring

EntireName: Cross-linked human gTuRC oligomeric ring
Components
  • Complex: Cross-linked human gTuRC oligomeric ring
    • Protein or peptide: GCP2
    • Protein or peptide: GCP3
    • Protein or peptide: gamma-tubulin

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Supramolecule #1: Cross-linked human gTuRC oligomeric ring

SupramoleculeName: Cross-linked human gTuRC oligomeric ring / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: GCP2

MacromoleculeName: GCP2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: MSEFRIHHDV NELLSLLRVH GGDGAEVYID LLQKNRTPYV TTTVSAHSAK VKIAEFSRTP EDFLKKYDE LKSKNTRNLD PLVYLLSKLT EDKETLQYLQ QNAKERAELA AAAVGSSTTS I NVPAAASK ISMQELEELR KQLGSVATGS TLQQSLELKR KMLRDKQNKK ...String:
MSEFRIHHDV NELLSLLRVH GGDGAEVYID LLQKNRTPYV TTTVSAHSAK VKIAEFSRTP EDFLKKYDE LKSKNTRNLD PLVYLLSKLT EDKETLQYLQ QNAKERAELA AAAVGSSTTS I NVPAAASK ISMQELEELR KQLGSVATGS TLQQSLELKR KMLRDKQNKK NSGQHLPIFP AW VYERPAL IGDFLIGAGI STDTALPIGT LPLASQESAV VEDLLYVLVG VDGRYVSAQP LAG RQSRTF LVDPNLDLSI RELVHRILPV AASYSAVTRF IEEKSSFEYG QVNHALAAAM RTLV KEHLI LVSQLEQLHR QGLLSLQKLW FYIQPAMRTM DILASLATSV DKGECLGGST LSLLH DRSF SYTGDSQAQE LCLYLTKAAS APYFEVLEKW IYRGIIHDPY SEFMVEEHEL RKERIQ EDY NDKYWDQRYT IVQQQIPSFL QKMADKILST GKYLNVVREC GHDVTCPVAK EIIYTLK ER AYVEQIEKAF NYASKVLLDF LMEEKELVAH LRSIKRYFLM DQGDFFVHFM DLAEEELR K PVEDITPPRL EALLELALRM STANTDPFKD DLKIDLMPHD LITQLLRVLA IETKQEKAM AHADPTELAL SGLEAFSFDY IVKWPLSLII NRKALTRYQM LFRHMFYCKH VERQLCSVWI SNKTAKQHS LHSAQWFAGA FTLRQRMLNF VQNIQYYMMF EVMEPTWHIL EKNLKSASNI D DVLGHHTG FLDTCLKDCM LTNPELLKVF SKLMSVCVMF TNCMQKFTQS MKLDGELGGQ TL EHSTVLG LPAGAEERAR KELARKHLAE HADTVQLVSG FEATINKFDK NFSAHLLDLL ARL SIYSTS DCEHGMASVI SRLDFNGFYT ERLERLSAER SQKATPQVPV LRGPPAPAPR VAVT AQ

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Macromolecule #2: GCP3

MacromoleculeName: GCP3 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: MATPDQKSPN VLLQNLCCRI LGRSEADVAQ QFQYAVRVIG SNFAPTVERD EFLVAEKIKK ELIRQRREA DAALFSELHR KLHSQGVLKN KWSILYLLLS LSEDPRRQPS KVSSYATLFA Q ALPRDAHS TPYYYARPQT LPLSYQDRSA QSAQSSGSVG SSGISSIGLC ...String:
MATPDQKSPN VLLQNLCCRI LGRSEADVAQ QFQYAVRVIG SNFAPTVERD EFLVAEKIKK ELIRQRREA DAALFSELHR KLHSQGVLKN KWSILYLLLS LSEDPRRQPS KVSSYATLFA Q ALPRDAHS TPYYYARPQT LPLSYQDRSA QSAQSSGSVG SSGISSIGLC ALSGPAPAPQ SL LPGQSNQ APGVGDCLRQ QLGSRLAWTL TANQPSSQAT TSKGVPSAVS RNMTRSRREG DTG GTMEIT EAALVRDILY VFQGIDGKNI KMNNTENCYK VEGKANLSRS LRDTAVRLSE LGWL HNKIR RYTDQRSLDR SFGLVGQSFC AALHQELREY YRLLSVLHSQ LQLEDDQGVN LGLES SLTL RRLLVWTYDP KIRLKTLAAL VDHCQGRKGG ELASAVHAYT KTGDPYMRSL VQHILS LVS HPVLSFLYRW IYDGELEDTY HEFFVASDPT VKTDRLWHDK YTLRKSMIPS FMTMDQS RK VLLIGKSINF LHQVCHDQTP TTKMIAVTKS AESPQDAADL FTDLENAFQG KIDAAYFE T SKYLLDVLNK KYSLLDHMQA MRRYLLLGQG DFIRHLMDLL KPELVRPATT LYQHNLTGI LETAVRATNA QFDSPEILRR LDVRLLEVSP GDTGWDVFSL DYHVDGPIAT VFTRECMSHY LRVFNFLWR AKRMEYILTD IRKGHMCNAK LLRNMPEFSG VLHQCHILAS EMVHFIHQMQ Y YITFEVLE CSWDELWNKV QQAQDLDHII AAHEVFLDTI ISRCLLDSDS RALLNQLRAV FD QIIELQN AQDAIYRAAL EELQRRLQFE EKKKQREIEG QWGVTAAEEE EENKRIGEFK ESI PKMCSQ LRILTHFYQG IVQQFLVLLT TSSDESLRFL SFRLDFNEHY KAREPRLRVS LGTR GRRSS HT

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Macromolecule #3: gamma-tubulin

MacromoleculeName: gamma-tubulin / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: MPREIITLQL GQCGNQIGFE FWKQLCAEHG ISPEGIVEEF ATEGTDRKDV FFYQADDEHY IPRAVLLDL EPRVIHSILN SPYAKLYNPE NIYLSEHGGG AGNNWASGFS QGEKIHEDIF D IIDREADG SDSLEGFVLC HSIAGGTGSG LGSYLLERLN DRYPKKLVQT ...String:
MPREIITLQL GQCGNQIGFE FWKQLCAEHG ISPEGIVEEF ATEGTDRKDV FFYQADDEHY IPRAVLLDL EPRVIHSILN SPYAKLYNPE NIYLSEHGGG AGNNWASGFS QGEKIHEDIF D IIDREADG SDSLEGFVLC HSIAGGTGSG LGSYLLERLN DRYPKKLVQT YSVFPNQDEM SD VVVQPYN SLLTLKRLTQ NADCVVVLDN TALNRIATDR LHIQNPSFSQ INQLVSTIMS AST TTLRYP GYMNNDLIGL IASLIPTPRL HFLMTGYTPL TTDQSVASVR KTTVLDVMRR LLQP KNVMV STGRDRQTNH CYIAILNIIQ GEVDPTQVHK SLQRIRERKL ANFIPWGPAS IQVAL SRKS PYLPSAHRVS GLMMANHTSI SSLFERTCRQ YDKLRKREAF LEQFRKEDMF KDNFDE MDT SREIVQQLID EYHAATRPDY ISWGTQEQ

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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: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 2368 / Average electron dose: 42.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
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: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 8.78 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 11120
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: cryoSPARC (ver. 4.0+), RELION (ver. 4.0))
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationSoftware - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7as4


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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