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- EMDB-51020: Structure of the Position 7 CMG-decorated gamma-Tubulin Ring Comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-51020
TitleStructure of the Position 7 CMG-decorated gamma-Tubulin Ring Complex from Pig Brain
Map data
Sample
  • Complex: Gamma-Tubulin Ring Complex in native pig brain
    • Protein or peptide: Gamma-tubulin complex component 3
    • Protein or peptide: Gamma-tubulin complex component
    • Protein or peptide: Mitotic-spindle organizing protein 2A isoform X4
    • Protein or peptide: CDK5 regulatory subunit-associated protein 2
KeywordsTubulin Complex / STRUCTURAL PROTEIN
Function / homology
Function and homology information


microtubule organizing center organization / Recruitment of mitotic centrosome proteins and complexes / negative regulation of centriole replication / equatorial microtubule organizing center / regulation of mitotic cell cycle spindle assembly checkpoint / polar microtubule / microtubule plus-end / gamma-tubulin complex / microtubule nucleation / microtubule bundle formation ...microtubule organizing center organization / Recruitment of mitotic centrosome proteins and complexes / negative regulation of centriole replication / equatorial microtubule organizing center / regulation of mitotic cell cycle spindle assembly checkpoint / polar microtubule / microtubule plus-end / gamma-tubulin complex / microtubule nucleation / microtubule bundle formation / gamma-tubulin binding / centrosome cycle / Recruitment of NuMA to mitotic centrosomes / regulation of neuron differentiation / pericentriolar material / mitotic spindle pole / establishment of mitotic spindle orientation / centriole replication / negative regulation of neuron differentiation / spindle assembly / cytoplasmic microtubule organization / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Recruitment of NuMA to mitotic centrosomes / positive regulation of microtubule polymerization / Anchoring of the basal body to the plasma membrane / centriole / AURKA Activation by TPX2 / tubulin binding / neurogenesis / meiotic cell cycle / chromosome segregation / neuron migration / brain development / microtubule cytoskeleton organization / spindle pole / spindle / Regulation of PLK1 Activity at G2/M Transition / cell junction / mitotic cell cycle / microtubule binding / microtubule / cytoskeleton / calmodulin binding / transcription cis-regulatory region binding / centrosome / protein-containing complex binding / protein kinase binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / Golgi apparatus / extracellular exosome / nucleoplasm / cytosol / cytoplasm
Similarity search - Function
CDK5 regulatory subunit-associated protein 2 / MOZART2 family / Mitotic-spindle organizing gamma-tubulin ring associated / Centrosomin, N-terminal motif 1 / Centrosomin N-terminal motif 1 / 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
Similarity search - Domain/homology
Gamma-tubulin complex component / Mitotic-spindle organizing protein 2A isoform X4 / Gamma-tubulin complex component 3 / CDK5 regulatory subunit-associated protein 2
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsMunoz-Hernandez H / Krutyholowa R / Wieczorek M
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science FoundationTMSGI3_211309 Switzerland
Swiss National Science Foundation310030_208120 Switzerland
CitationJournal: Dev Cell / Year: 2024
Title: Partial closure of the γ-tubulin ring complex by CDK5RAP2 activates microtubule nucleation.
Authors: Yixin Xu / Hugo Muñoz-Hernández / Rościsław Krutyhołowa / Florina Marxer / Ferdane Cetin / Michal Wieczorek /
Abstract: Microtubule nucleation is templated by the γ-tubulin ring complex (γ-TuRC), but its structure deviates from the geometry of α-/β-tubulin in the microtubule, explaining the complex's poor ...Microtubule nucleation is templated by the γ-tubulin ring complex (γ-TuRC), but its structure deviates from the geometry of α-/β-tubulin in the microtubule, explaining the complex's poor nucleating activity. Several proteins may activate the γ-TuRC, but the mechanisms underlying activation are not known. Here, we determined the structure of the porcine γ-TuRC purified using CDK5RAP2's centrosomin motif 1 (CM1). We identified an unexpected conformation of the γ-TuRC bound to multiple protein modules containing MZT2, GCP2, and CDK5RAP2, resulting in a long-range constriction of the γ-tubulin ring that brings it in closer agreement with the 13-protofilament microtubule. Additional CDK5RAP2 promoted γ-TuRC decoration and stimulated the microtubule-nucleating activities of the porcine γ-TuRC and a reconstituted, CM1-free human complex in single-molecule assays. Our results provide a structural mechanism for the control of microtubule nucleation by CM1 proteins and identify conformational transitions in the γ-TuRC that prime it for microtubule nucleation.
History
DepositionJul 12, 2024-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51020.png

Downloads & links

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Map

FileDownload / File: emd_51020.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.41 Å/pix.
x 320 pix.
= 452.266 Å		1.41 Å/pix.
x 320 pix.
= 452.266 Å		1.41 Å/pix.
x 320 pix.
= 452.266 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.41333 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0075
Minimum - Maximum-0.010181445 - 0.0344795
Average (Standard dev.)0.000031672207 (±0.00077062694)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 452.2656 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_51020_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_51020_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Gamma-Tubulin Ring Complex in native pig brain

EntireName: Gamma-Tubulin Ring Complex in native pig brain
Components
  • Complex: Gamma-Tubulin Ring Complex in native pig brain
    • Protein or peptide: Gamma-tubulin complex component 3
    • Protein or peptide: Gamma-tubulin complex component
    • Protein or peptide: Mitotic-spindle organizing protein 2A isoform X4
    • Protein or peptide: CDK5 regulatory subunit-associated protein 2

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Supramolecule #1: Gamma-Tubulin Ring Complex in native pig brain

SupramoleculeName: Gamma-Tubulin Ring Complex in native pig brain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Sus scrofa (pig)

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Macromolecule #1: Gamma-tubulin complex component 3

MacromoleculeName: Gamma-tubulin complex component 3 / type: protein_or_peptide / ID: 1 / Details: SsGCP3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
Molecular weightTheoretical: 103.172477 KDa
SequenceString: MATPDQKSPN VLLQNLCCRI LGKSEADVAQ QFQYAVRVIG SNFAPTVERD EFLVAEKIKK ELTRQRREAD AALFSELHRK LHSQGVLKN KWSILYLLLS LSEDPRKQPS KVSGYAALFA QALPRDAHST PYYYARPQSL PLNYQERGAP SAQSAGSAGS S GVSSLGTY ...String:
MATPDQKSPN VLLQNLCCRI LGKSEADVAQ QFQYAVRVIG SNFAPTVERD EFLVAEKIKK ELTRQRREAD AALFSELHRK LHSQGVLKN KWSILYLLLS LSEDPRKQPS KVSGYAALFA QALPRDAHST PYYYARPQSL PLNYQERGAP SAQSAGSAGS S GVSSLGTY ALNGPTPPPP PPALLPGQPL PAPGVGDGLR QQLGSRLAWT LTASQPSLPS TTSKAVPSSG SRGAARPRRE GD AAAGAVE VTEAALVRDI LYVFQGIDGK HVKMSNADNC YTVEGKANLS KSLRDTAVRL AELGWLHNKI RKYTDQRSLD RSF GLVGQS FCAALHQELR EYYRLLSVLH SQLQLEDDQG VNLGLESSLT LRRLLVWTYD PKMRLKTLAA LVDHCQGRKG GELA SAVHA YTKTGDPYAR SLVQHILSLV SHPVLSFLYR WIYDGELEDT YHEFFVASDP AVKADRLWHD KYALRKPMIP SFMTM DQCR KVLLIGKSIN FLHQVCHDQT PTTKMIAVTK SAESPQDAAD LFTDLENAFQ GKIDAAYFET SKYLLDVLNK KYSLLD HMQ AMRRYLLLGQ GDFIRHLMDL LKPELVRPAT TLYQHNLTGI LETAVRATNA QFDSPEILKR LDVRLLEVSP GDTGWDV FS LDYHVDGPIA TVFTRECMSH YLRAFNFLWR AKRVEYILTD IRKGHMCNAR LLRSMPEFSG VLHHCHILAS EMVHFIHQ M QYYVTFEVLE CSWDELWNRV QRAQDLDHII AAHEAFLGTV ISRCLLDSDS RALLNQLRAV FDQIIELQNT QDAIYRAAL EELQRRLQFE EKKKQREAEG QWGVSAAEEE QEKRRVQEFQ ESIPKMCSQL RILTHFYQGV VQQFLVSLTT SSDESLRFLS FRLDFNEHY RAREPRLRVS LGTRGRRSSH T

UniProtKB: Gamma-tubulin complex component 3

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Macromolecule #2: Gamma-tubulin complex component

MacromoleculeName: Gamma-tubulin complex component / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
Molecular weightTheoretical: 102.609703 KDa
SequenceString: MSEFRIHHDV NELLSLLRVH GGDGAEVYID LLQKNRTPYV TTTVSAHSAK VKIAEFSRTP EDFLKKYDEL KSKNTRNLDP LVYLLSKLM EDRETLQYLQ QNAKERAELA ASAAASSTAS FGASATASKI SMQELEELRK QLGSVATGPT WQQSLELTRK M LRDKQSKK ...String:
MSEFRIHHDV NELLSLLRVH GGDGAEVYID LLQKNRTPYV TTTVSAHSAK VKIAEFSRTP EDFLKKYDEL KSKNTRNLDP LVYLLSKLM EDRETLQYLQ QNAKERAELA ASAAASSTAS FGASATASKI SMQELEELRK QLGSVATGPT WQQSLELTRK M LRDKQSKK NSGQRLPVLP AWVYERPALL GDFLPGTGGS ADTAVPIGSL PLASQEAAVV EDLLYVLVGV DGRYISAQPL TG RQGRTFL VDPNLDLSIR ELVSRILPVA ASYSTVTRFI EEKSSFEYGQ VNHALAAAMR TLVKEYLVLV TQLEQLQRQG LLS LQKLWF YIQPAMRSLD ILASLATSVD KGECIGGATL SLLHDRSFSY TGDSQAQELC LHLTKAASTP YFEILEKWIY RGII DDPYS EFMVEEHELR KEKIQEDYND KYWDQRYTVV QRQIPSFLQK MAGKVLSTGK YLNVVRECGH DVTCPVAKEV VYTLK ERAY VEQIEKAFSY ASKVLLDFLM GEKELLAHLR SIKRYFLMDQ GDFFVHFMDL TEEELKKPVD DITPTRLEAL LELALR MST ANTDPFKDDL KIDLMPHDLI TQLLRVLAIE TQQEKAMVHA DPTELTLSGL EAFSFDYVVT WPLSLIINRK ALTRYQM LF RHMFYCKHVE RQLCSVWISN KAAKRFSLHS AKWFAGAFTL RQRMLNFVQN IQSYMMFEVM EPTWHVLEQN LRSASNID D VLGHHASFLD NCLKDCMLTN PELLRVFSKL MSVCVMFTNC LQRFTQSMKL DSELGHPALE PGAMLGPPTE AERAEERAR KELARKCLAE HVDAPQLASS FEATITKFDK NFSAHLLDLL ARLSIYSTSD CEHGMASVIS RLDFNGFYTE RLERLSAERS QKAAPPVPG PRGPPALVPR VAVTAQ

UniProtKB: Gamma-tubulin complex component

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Macromolecule #3: Mitotic-spindle organizing protein 2A isoform X4

MacromoleculeName: Mitotic-spindle organizing protein 2A isoform X4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
Molecular weightTheoretical: 15.920321 KDa
SequenceString:
MAAPGAGPGP GAPPGLEAAL QKLALRRKKV LSAEETELFE LAQAAGGAMD PEVFKILVDL LKLNVAPLAV FQMLKSMCAG QRLASEPQD PVAVPLPTTS VPETRGRNRG SSALGGGPAL AERSGREGSS QRMPRQPSAT RLPKGGGPGK SPTRST

UniProtKB: Mitotic-spindle organizing protein 2A isoform X4

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Macromolecule #4: CDK5 regulatory subunit-associated protein 2

MacromoleculeName: CDK5 regulatory subunit-associated protein 2 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 215.344219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MMDLVLEEDV TVPGTLSGCS GLVPSVPDDL DGINPNAGLG NGLLPNVSEE TVSPTRARNM KDFENQITEL KKENFNLKLR IYFLEERMQ QEFHGPTEHI YKTNIELKVE VESLKRELQE REQLLIKASK AVESLAEAGG SEIQRVKEDA RKKVQQVEDL L TKRILLLE ...String:
MMDLVLEEDV TVPGTLSGCS GLVPSVPDDL DGINPNAGLG NGLLPNVSEE TVSPTRARNM KDFENQITEL KKENFNLKLR IYFLEERMQ QEFHGPTEHI YKTNIELKVE VESLKRELQE REQLLIKASK AVESLAEAGG SEIQRVKEDA RKKVQQVEDL L TKRILLLE KDVTAAQAEL EKAFAGTETE KALRLRLESK LSEMKKMHEG DLAMALVLDE KDRLIEELKL SLKSKEALIQ CL KEEKSQM ACPDENVSSG ELRGLCAAPR EEKERETEAA QMEHQKERNS FEERIQALEE DLREKEREIA TEKKNSLKRD KAI QGLTMA LKSKEKKVEE LNSEIEKLSA AFAKAREALQ KAQTQEFQGS EDYETALSGK EALSAALRSQ NLTKSTENHR LRRS IKKIT QELSDLQQER ERLEKDLEEA HREKSKGDCT IRDLRNEVEK LRNEVNEREK AMENRYKSLL SESNKKLHNQ EQVIK HLTE STNQKDVLLQ KFNEKDLEVI QQNCYLMAAE DLELRSEGLI TEKCSSQQPP GSKTIFSKEK KQSSDYEELI QVLKKE QDI YTHLVKSLQE SDSINNLQAE LNKIFALRKQ LEQDVLSYQN LRKTLEEQIS EIRRREEESF SLYSDQTSYL SICLEEN NR FQVEHFSQEE LKKKVSDLIQ LVKELYTDNQ HLKKTIFDLS CMGFQGNGFP DRLASTEQTE LLASKEDEDT IKIGEDDE I NFLSDQHLQQ SNEIMKDLSK GGCKNGYLRH TESKISDCDG AHAPGCLEEG AFINLLAPLF NEKATLLLES RPDLLKVVR ELLLGQLFLT EQEVSGEHLD GKTEKTPKQK GELVHFVQTN SFSKPHDELK LSCEAQLVKA GEVPKVGLKD ASVQTVATEG DLLRFKHEA TREAWEEKPI NTALSAEHRP ENLHGVPGWQ AALLSLPGIT NREAKKSRLP ILIKPSRSLG NMYRLPATQE V VTQLQSQI LELQGELKEF KTCNKQLHQK LILAEAVMEG RPTPDKTLLN AQPPVGAAYQ DSPGEQKGIK TTSSVWRDKE MD SDQQRSY EIDSEICPPD DLASLPSCKE NPEDVLSPTS VATYLSSKSQ PSAKVSVMGT DQSESINTSN ETEYLKQKIH DLE TELEGY QNFIFQLQKH SQCSEAIITV LCGTEGAQDG LSKPKNGSDG EEMTFSSLHQ VRYVKHVKIL GPLAPEMIDS RVLE NLKQQ LEEQEYKLQK EQNLNMQLFS EIHNLQNKFR DLSPPRYDSL VQSQARELSL QRQQIKDGHG ICVISRQHMN TMIKA FEEL LQASDVDYCV AEGFQEQLNQ CAELLEKLEK LFLNGKSVGV EMNTQNELME RIEEDNLTYQ HLLPESPEPS ASHALS DYE TSEKSFFSRD QKQDNETEKT SVMVNSFSQD LLMEHIQEIR TLRKRLEESI KTNEKLRKQL ERQGSEFVQG STSIFAS GS ELHSSLTSEI HFLRKQNQAL NAMLIKGSRD KQKENDKLRE SLSRKTVSLE HLQREYASVK EENERLQKEG SEKERHNQ Q LIQEVRCSGQ ELSRVQEEVK LRQQLLSQND KLLQSLRVEL KAYEKLDEEH RRLREASGEG WKGQDPFRDL HSLLMEIQA LRLQLERSIE TSSTLQSRLK EQLARGAEKA QEGALTLAVQ AVSIPEVPLQ PDKHDGDKYP MESDNSFDLF DSSQAVTPKS VSETPPLSG NDTDSLSCDS GSSATSTPCV SRLVTGHHLW ASKNGRHVLG LIEDYEALLK QISQGQRLLA EMDIQTQEAP S STSQELGT KGPHPAPLSK FVSSVSTAKL TLEEAYRRLK LLWRVSLPED GQCPLHCEQI GEMKAEVTKL HKKLFEQEKK LQ NTMKLLQ LSKRQEKVIF DQLVVTHKIL RKARGNLELR PGGAHPGTCS PSRPGS

UniProtKB: CDK5 regulatory subunit-associated protein 2

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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
Component:
ConcentrationName
40.0 mMHEPES
150.0 mMNaCl
1.0 mMMgCl2
1.0 mMEGTA
2.0 mM2-mercaptoethanol
0.01 % v/vIGEPAL
0.1 mMGTP

Details: Listed in "g-TuRC cryo-EM sample preparation" section.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.9 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93207
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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