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- EMDB-53399: Cryo-EM reconstruction of the NEDD1 anchor protein and CDK5RAP2 b... -

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Basic information

Entry
Database: EMDB / ID: EMD-53399
TitleCryo-EM reconstruction of the NEDD1 anchor protein and CDK5RAP2 bound to the gamma-tubulin ring complex
Map data
Sample
  • Complex: Cryo-EM reconstruction of the NEDD1 and CDK5RAP2 bound to the gamma-tubulin ring complex
    • Protein or peptide: x 11 types
KeywordsTubulin complex / STRUCTURAL PROTEIN
Function / homology
Function and homology information


microtubule nucleation by interphase microtubule organizing center / gamma-tubulin complex localization / microtubule nucleator activity / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / polar microtubule / interphase microtubule organizing center / bBAF complex / gamma-tubulin complex / gamma-tubulin ring complex ...microtubule nucleation by interphase microtubule organizing center / gamma-tubulin complex localization / microtubule nucleator activity / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / polar microtubule / interphase microtubule organizing center / bBAF complex / gamma-tubulin complex / gamma-tubulin ring complex / npBAF complex / regulation of transepithelial transport / nBAF complex / mitotic spindle microtubule / brahma complex / meiotic spindle organization / morphogenesis of a polarized epithelium / protein localization to adherens junction / postsynaptic actin cytoskeleton / structural constituent of postsynaptic actin cytoskeleton / Formation of the dystrophin-glycoprotein complex (DGC) / GBAF complex / Formation of annular gap junctions / Tat protein binding / Gap junction degradation / regulation of G0 to G1 transition / Folding of actin by CCT/TriC / Cell-extracellular matrix interactions / dense body / microtubule nucleation / regulation of nucleotide-excision repair / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / apical protein localization / regulation of double-strand break repair / gamma-tubulin binding / adherens junction assembly / RHOF GTPase cycle / Adherens junctions interactions / non-motile cilium / tight junction / Sensory processing of sound by outer hair cells of the cochlea / Interaction between L1 and Ankyrins / SWI/SNF complex / regulation of mitotic metaphase/anaphase transition / Sensory processing of sound by inner hair cells of the cochlea / positive regulation of T cell differentiation / regulation of norepinephrine uptake / transporter regulator activity / apical junction complex / nitric-oxide synthase binding / positive regulation of double-strand break repair / pericentriolar material / maintenance of blood-brain barrier / NuA4 histone acetyltransferase complex / establishment or maintenance of cell polarity / cortical cytoskeleton / cell leading edge / positive regulation of stem cell population maintenance / Regulation of MITF-M-dependent genes involved in pigmentation / microtubule organizing center / Recycling pathway of L1 / regulation of synaptic vesicle endocytosis / regulation of G1/S transition of mitotic cell cycle / brush border / mitotic sister chromatid segregation / kinesin binding / EPH-ephrin mediated repulsion of cells / negative regulation of cell differentiation / single fertilization / mitotic spindle assembly / RHO GTPases Activate WASPs and WAVEs / positive regulation of myoblast differentiation / RHO GTPases activate IQGAPs / positive regulation of double-strand break repair via homologous recombination / regulation of protein localization to plasma membrane / cytoplasmic microtubule / spindle assembly / cytoplasmic microtubule organization / cytoskeleton organization / EPHB-mediated forward signaling / substantia nigra development / centriole / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / calyx of Held / Recruitment of mitotic centrosome proteins and complexes / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / axonogenesis / AURKA Activation by TPX2 / condensed nuclear chromosome / mitotic spindle organization / meiotic cell cycle / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / adherens junction / positive regulation of cell differentiation / actin filament / FCGR3A-mediated phagocytosis
Similarity search - Function
: / CDK5 regulatory subunit-associated protein 2 / : / CDK5 regulatory subunit-associated protein 2-like, coiled coil / MOZART2 family / Mitotic-spindle organizing gamma-tubulin ring associated / Centrosomin, N-terminal motif 1 / Centrosomin N-terminal motif 1 / Mitotic-spindle organizing protein 1 / Mitotic-spindle organizing gamma-tubulin ring associated ...: / CDK5 regulatory subunit-associated protein 2 / : / CDK5 regulatory subunit-associated protein 2-like, coiled coil / MOZART2 family / Mitotic-spindle organizing gamma-tubulin ring associated / Centrosomin, N-terminal motif 1 / Centrosomin N-terminal motif 1 / Mitotic-spindle organizing protein 1 / Mitotic-spindle organizing gamma-tubulin ring associated / Gamma-tubulin complex component 6, N-terminal / Gamma-tubulin complex component 6 N-terminus / Gamma tubulin / 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 / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Actin / Actin family / Actin / 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 / ATPase, nucleotide binding domain / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
CDK5 regulatory subunit-associated protein 2 / TUBGCP6 protein / Tubulin gamma-1 chain / Actin, cytoplasmic 1 / Mitotic-spindle organizing protein 1 / Mitotic-spindle organizing protein 2B / Protein NEDD1 / Gamma-tubulin complex component 3 / Gamma-tubulin complex component 5 / Gamma-tubulin complex component 2 / Gamma-tubulin complex component 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsMunoz-Hernandez H / Xu Y / Wieczorek M
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_208120 Switzerland
Swiss National Science FoundationTMSGI3_211309 Switzerland
CitationJournal: bioRxiv / Year: 2024
Title: Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex.
Authors: Hugo Muñoz-Hernández / Yixin Xu / Daniel Zhang / Allen Xue / Amol Aher / Aitor Pellicer Camardiel / Ellie Walker / Florina Marxer / Tarun M Kapoor / Michal Wieczorek /
Abstract: The γ-tubulin ring complex (γ-TuRC) is an essential multiprotein assembly, in which γ-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for ...The γ-tubulin ring complex (γ-TuRC) is an essential multiprotein assembly, in which γ-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The γ-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the γ-TuRC. However, the structural basis of the NEDD1-γ-TuRC interaction is not known. Here, we report cryo-electron microscopy (cryo-EM) structures of NEDD1 bound to the human γ-TuRC in the absence or presence of the activating factor CDK5RAP2, which interacts with GCP2 to induce conformational changes in the γ-TuRC and promote its microtubule nucleating function. We found that the C-terminus of NEDD1 forms a tetrameric α-helical assembly that contacts the lumen of the γ-TuRC cone, is anchored to GCP4, 5 and 6 via protein modules consisting of MZT1 & GCP3 subcomplexes, and orients its microtubule-binding WD40 domains away from the complex. We biochemically tested our structural models by identifying NEDD1 mutants unable to pull-down -tubulin from cultured cells. The structure of the γ-TuRC simultaneously bound to NEDD1 and CDK5RAP2 reveals that both factors can associate with the "open" conformation of the complex. Our results show that NEDD1 does not induce conformational changes in the γ-TuRC, but suggest that anchoring of γ-TuRC-capped microtubules by NEDD1 would be structurally compatible with the significant conformational changes experienced by the γ-TuRC during microtubule nucleation.
History
DepositionApr 11, 2025-
Header (metadata) releaseMay 21, 2025-
Map releaseMay 21, 2025-
UpdateJun 4, 2025-
Current statusJun 4, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53399.png

Downloads & links

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Map

FileDownload / File: emd_53399.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.41 Å/pix.
x 384 pix.
= 541.44 Å		1.41 Å/pix.
x 384 pix.
= 541.44 Å		1.41 Å/pix.
x 384 pix.
= 541.44 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.41 Å
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Contour LevelBy AUTHOR: 0.0363
Minimum - Maximum-0.07280999 - 0.30975085
Average (Standard dev.)0.0020313493 (±0.015650984)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 541.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_53399_additional_1.map
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Half map: #1

Fileemd_53399_half_map_1.map
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Half map: #2

Fileemd_53399_half_map_2.map
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Sample components

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Entire : Cryo-EM reconstruction of the NEDD1 and CDK5RAP2 bound to the gam...

EntireName: Cryo-EM reconstruction of the NEDD1 and CDK5RAP2 bound to the gamma-tubulin ring complex
Components
  • Complex: Cryo-EM reconstruction of the NEDD1 and CDK5RAP2 bound to the gamma-tubulin ring complex
    • Protein or peptide: Gamma-tubulin complex component 3
    • Protein or peptide: Mitotic-spindle organizing protein 1
    • Protein or peptide: Protein NEDD1
    • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
    • Protein or peptide: Tubulin gamma-1 chain
    • Protein or peptide: Gamma-tubulin complex component 4
    • Protein or peptide: Isoform 3 of Gamma-tubulin complex component 2
    • Protein or peptide: TUBGCP6 protein
    • Protein or peptide: Gamma-tubulin complex component 5
    • Protein or peptide: Mitotic-spindle organizing protein 2B
    • Protein or peptide: CDK5 regulatory subunit-associated protein 2

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Supramolecule #1: Cryo-EM reconstruction of the NEDD1 and CDK5RAP2 bound to the gam...

SupramoleculeName: Cryo-EM reconstruction of the NEDD1 and CDK5RAP2 bound to the gamma-tubulin ring complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Gamma-tubulin complex component 3

MacromoleculeName: Gamma-tubulin complex component 3 / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 103.710102 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MATPDQKSPN VLLQNLCCRI LGRSEADVAQ QFQYAVRVIG SNFAPTVERD EFLVAEKIKK ELIRQRREAD AALFSELHRK LHSQGVLKN KWSILYLLLS LSEDPRRQPS KVSSYATLFA QALPRDAHST PYYYARPQTL PLSYQDRSAQ SAQSSGSVGS S GISSIGLC ...String:
MATPDQKSPN VLLQNLCCRI LGRSEADVAQ QFQYAVRVIG SNFAPTVERD EFLVAEKIKK ELIRQRREAD AALFSELHRK LHSQGVLKN KWSILYLLLS LSEDPRRQPS KVSSYATLFA QALPRDAHST PYYYARPQTL PLSYQDRSAQ SAQSSGSVGS S GISSIGLC ALSGPAPAPQ SLLPGQSNQA PGVGDCLRQQ LGSRLAWTLT ANQPSSQATT SKGVPSAVSR NMTRSRREGD TG GTMEITE AALVRDILYV FQGIDGKNIK MNNTENCYKV EGKANLSRSL RDTAVRLSEL GWLHNKIRRY TDQRSLDRSF GLV GQSFCA ALHQELREYY RLLSVLHSQL QLEDDQGVNL GLESSLTLRR LLVWTYDPKI RLKTLAALVD HCQGRKGGEL ASAV HAYTK TGDPYMRSLV QHILSLVSHP VLSFLYRWIY DGELEDTYHE FFVASDPTVK TDRLWHDKYT LRKSMIPSFM TMDQS RKVL LIGKSINFLH QVCHDQTPTT KMIAVTKSAE SPQDAADLFT DLENAFQGKI DAAYFETSKY LLDVLNKKYS LLDHMQ AMR RYLLLGQGDF IRHLMDLLKP ELVRPATTLY QHNLTGILET AVRATNAQFD SPEILRRLDV RLLEVSPGDT GWDVFSL DY HVDGPIATVF TRECMSHYLR VFNFLWRAKR MEYILTDIRK GHMCNAKLLR NMPEFSGVLH QCHILASEMV HFIHQMQY Y ITFEVLECSW DELWNKVQQA QDLDHIIAAH EVFLDTIISR CLLDSDSRAL LNQLRAVFDQ IIELQNAQDA IYRAALEEL QRRLQFEEKK KQREIEGQWG VTAAEEEEEN KRIGEFKESI PKMCSQLRIL THFYQGIVQQ FLVLLTTSSD ESLRFLSFRL DFNEHYKAR EPRLRVSLGT RGRRSSHT

UniProtKB: Gamma-tubulin complex component 3

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Macromolecule #2: Mitotic-spindle organizing protein 1

MacromoleculeName: Mitotic-spindle organizing protein 1 / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.485724 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MASSSGAGAA AAAAAANLNA VRETMDVLLE ISRILNTGLD METLSICVRL CEQGINPEAL SSVIKELRKA TEALKAAENM TS

UniProtKB: Mitotic-spindle organizing protein 1

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Macromolecule #3: Protein NEDD1

MacromoleculeName: Protein NEDD1 / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 72.050984 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MQENLRFASS GDDIKIWDAS SMTLVDKFNP HTSPHGISSI CWSSNNNFLV TASSSGDKIV VSSCKCKPVP LLELAEGQKQ TCVNLNSTS MYLVSGGLNN TVNIWDLKSK RVHRSLKDHK DQVTCVTYNW NDCYIASGSL SGEIILHSVT TNLSSTPFGH G SNQSVRHL ...String:
MQENLRFASS GDDIKIWDAS SMTLVDKFNP HTSPHGISSI CWSSNNNFLV TASSSGDKIV VSSCKCKPVP LLELAEGQKQ TCVNLNSTS MYLVSGGLNN TVNIWDLKSK RVHRSLKDHK DQVTCVTYNW NDCYIASGSL SGEIILHSVT TNLSSTPFGH G SNQSVRHL KYSLFKKSLL GSVSDNGIVT LWDVNSQSPY HNFDSVHKAP ASGICFSPVN ELLFVTIGLD KRIILYDTSS KK LVKTLVA DTPLTAVDFM PDGATLAIGS SRGKIYQYDL RMLKSPVKTI SAHKTSVQCI AFQYSTVLTK SSLNKGCSNK PTT VNKRSV NVNAASGGVQ NSGIVREAPA TSIATVLPQP MTSAMGKGTV AVQEKAGLPR SINTDTLSKE TDSGKNQDFS SFDD TGKSS LGDMFSPIRD DAVVNKGSDE SIGKGDGFDF LPQLNSVFPP RKNPVTSSTS VLHSSPLNVF MGSPGKEENE NRDLT AESK KIYMGKQESK DSFKQLAKLV TSGAESGNLN TSPSSNQTRN SEKFEKPENE IEAQLICEPP INGSSTPNPK IASSVT AGV ASSLSEKIAD SIGNNRQNAP LTSIQIRFIQ NMIQETLDDF REACHRDIVN LQVEMIKQFH MQLNEMHSLL ERYSVNE GL VAEIERLREE NKRLRAHF

UniProtKB: Protein NEDD1

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Macromolecule #4: Actin, cytoplasmic 1, N-terminally processed

MacromoleculeName: Actin, cytoplasmic 1, N-terminally processed / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.78266 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIVTNWD DMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSGDGV T HTVPIYEG ...String:
MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIVTNWD DMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSGDGV T HTVPIYEG YALPHAILRL DLAGRDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSLEK SY ELPDGQV ITIGNERFRC PEALFQPSFL GMESCGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKEI TAL APSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #5: Tubulin gamma-1 chain

MacromoleculeName: Tubulin gamma-1 chain / type: protein_or_peptide / ID: 5 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.022617 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MPREIITLQL GQCGNQIGFE FWKQLCAEHG ISPEGIVEEF ATEGTDRKDV FFYQADDEHY IPRAVLLDLE PRVIHSILNS PYAKLYNPE NIYLSEHGGG AGNNWASGFS QGEKIHEDIF DIIDREADGS DSLEGFVLCH SIAGGTGSGL GSYLLERLND R YPKKLVQT ...String:
MPREIITLQL GQCGNQIGFE FWKQLCAEHG ISPEGIVEEF ATEGTDRKDV FFYQADDEHY IPRAVLLDLE PRVIHSILNS PYAKLYNPE NIYLSEHGGG AGNNWASGFS QGEKIHEDIF DIIDREADGS DSLEGFVLCH SIAGGTGSGL GSYLLERLND R YPKKLVQT YSVFPNQDEM SDVVVQPYNS LLTLKRLTQN ADCVVVLDNT ALNRIATDRL HIQNPSFSQI NQLVSTIMSA ST TTLRYPG YMNNDLIGLI ASLIPTPRLH FLMTGYTPLT TDQSVASVRK TTVLDVMRRL LQPKNVMVST GRDRQTNHCY IAI LNIIQG EVDPTQVHKS LQRIRERKLA NFIPWGPASI QVALSRKSPY LPSAHRVSGL MMANHTSISS LFERTCRQYD KLRK REAFL EQFRKEDMFK DNFDEMDTSR EIVQQLIDEY HAATRPDYIS WGTQEQENLY FQ

UniProtKB: Tubulin gamma-1 chain

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Macromolecule #6: Gamma-tubulin complex component 4

MacromoleculeName: Gamma-tubulin complex component 4 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 76.179969 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MIHELLLALS GYPGSIFTWN KRSGLQVSQD FPFLHPSETS VLNRLCRLGT DYIRFTEFIE QYTGHVQQQD HHPSQQGQGG LHGIYLRAF CTGLDSVLQP YRQALLDLEQ EFLGDPHLSI SHVNYFLDQF QLLFPSVMVV VEQIKSQKIH GCQILETVYK H SCGGLPPV ...String:
MIHELLLALS GYPGSIFTWN KRSGLQVSQD FPFLHPSETS VLNRLCRLGT DYIRFTEFIE QYTGHVQQQD HHPSQQGQGG LHGIYLRAF CTGLDSVLQP YRQALLDLEQ EFLGDPHLSI SHVNYFLDQF QLLFPSVMVV VEQIKSQKIH GCQILETVYK H SCGGLPPV RSALEKILAV CHGVMYKQLS AWMLHGLLLD QHEEFFIKQG PSSGNVSAQP EEDEEDLGIG GLTGKQLREL QD LRLIEEE NMLAPSLKQF SLRVEILPSY IPVRVAEKIL FVGESVQMFE NQNVNLTRKG SILKNQEDTF AAELHRLKQQ PLF SLVDFE QVVDRIRSTV AEHLWKLMVE ESDLLGQLKI IKDFYLLGRG ELFQAFIDTA QHMLKTPPTA VTEHDVNVAF QQSA HKVLL DDDNLLPLLH LTIEYHGKEH KADATQAREG PSRETSPREA PASGWAALGL SYKVQWPLHI LFTPAVLEKY NVVFK YLLS VRRVQAELQH CWALQMQRKH LKSNQTDAIK WRLRNHMAFL VDNLQYYLQV DVLESQFSQL LHQINSTRDF ESIRLA HDH FLSNLLAQSF ILLKPVFHCL NEILDLCHSF CSLVSQNLGP LDERGAAQLS ILVKGFSRQS SLLFKILSSV RNHQINS DL AQLLLRLDYN KYYTQAGGTL GSFGM

UniProtKB: Gamma-tubulin complex component 4

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Macromolecule #7: Isoform 3 of Gamma-tubulin complex component 2

MacromoleculeName: Isoform 3 of Gamma-tubulin complex component 2 / type: protein_or_peptide / ID: 7 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 105.765719 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSEFRIHHDV NELLSLLRVH GGDGAEVYID LLQKNRTPYV TTTVSAHSAK VKIAEFSRTP EDFLKKYDEL KSKNTRNLDP LVYLLSKLT EDKETLQYLQ QNAKERAELA AAAVGSSTTS INVPAAASKI SMQELEELRK QLGSVATGST LQQSLELKRK M LRDKQNKK ...String:
MSEFRIHHDV NELLSLLRVH GGDGAEVYID LLQKNRTPYV TTTVSAHSAK VKIAEFSRTP EDFLKKYDEL KSKNTRNLDP LVYLLSKLT EDKETLQYLQ QNAKERAELA AAAVGSSTTS INVPAAASKI SMQELEELRK QLGSVATGST LQQSLELKRK M LRDKQNKK NSGQHLPIFP AWVYERPALI GDFLIGAGIS TDTALPIVLL RWNLALSPRL KCSGVISAHC NLHLPGTLPL AS QESAVVE DLLYVLVGVD GRYVSAQPLA GRQSRTFLVD PNLDLSIREL VHRILPVAAS YSAVTRFIEE KSSFEYGQVN HAL AAAMRT LVKEHLILVS QLEQLHRQGL LSLQKLWFYI QPAMRTMDIL ASLATSVDKG ECLGGSTLSL LHDRSFSYTG DSQA QELCL YLTKAASAPY FEVLEKWIYR GIIHDPYSEF MVEEHELRKE RIQEDYNDKY WDQRYTIVQQ QIPSFLQKMA DKILS TGKY LNVVRECGHD VTCPVAKEII YTLKERAYVE QIEKAFNYAS KVLLDFLMEE KELVAHLRSI KRYFLMDQGD FFVHFM DLA EEELRKPVED ITPPRLEALL ELALRMSTAN TDPFKDDLKI DLMPHDLITQ LLRVLAIETK QEKAMAHADP TELALSG LE AFSFDYIVKW PLSLIINRKA LTRYQMLFRH MFYCKHVERQ LCSVWISNKT AKQHSLHSAQ WFAGAFTLRQ RMLNFVQN I QYYMMFEVME PTWHILEKNL KSASNIDDVL GHHTGFLDTC LKDCMLTNPE LLKVFSKLMS VCVMFTNCMQ KFTQSMKLD GELGGQTLEH STVLGLPAGA EERARKELAR KHLAEHADTV QLVSGFEATI NKFDKNFSAH LLDLLARLSI YSTSDCEHGM ASVISRLDF NGFYTERLER LSAERSQKAT PQVPVLRGPP APAPRVAVTA Q

UniProtKB: Gamma-tubulin complex component 2

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Macromolecule #8: TUBGCP6 protein

MacromoleculeName: TUBGCP6 protein / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 199.732516 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MASITQLFDD LCEALLPAAK THLGQRSVNR KRAKRSLKKV AYNALFTNLF QDETQQLQPD MSKLPARNKI LMLSFDLRVG GLGPKADRL EELVEELEAA PCCPLLEVGS VLDLLVQLAG SGPPQVLPRK RDYFLNNKHV GRNVPYSGYD CDDLSVFEMD V QSLISREE ...String:
MASITQLFDD LCEALLPAAK THLGQRSVNR KRAKRSLKKV AYNALFTNLF QDETQQLQPD MSKLPARNKI LMLSFDLRVG GLGPKADRL EELVEELEAA PCCPLLEVGS VLDLLVQLAG SGPPQVLPRK RDYFLNNKHV GRNVPYSGYD CDDLSVFEMD V QSLISREE CLCHSMIQET LQVMEAAPGT GLPTVGLFSF GDPCGDRFER DTRVSLFGAL VHSRTYDMDV RLGLPPVPDN AD LSGLAIK VPPSVDQWED EGFQSASNLT PDSQSEPSVT PDVDLWEAAL TYEASKRRCW ERVGCPPGHR EEPYLTEAGR DAF DKFCRL HQGELQLLAG GVLQAPQPVL VKECELVKDV LNVLIGVVSA TFSLCQPAQA FVVKRGVHVS GASPESISSL LSEV AEYGT CYTRLSHFSL QPVLDSLYSK GLVFQAFTSG LRRYLQYYRA CVLSTPPTLS LLTIGFLFKK LGRQLRYLAE LCGVG AVLP GTCGGGPRAA FPTGVKLLSY LYQEALHNCS NEHYPVLLSL LKTSCEPYTR FIHDWVYSGV FRDAYGEFMI QVNHEY LSF RDKLYWTHGY VLISKEVEDC VPVFLKHIAH DIYVCGKTIN LLKLCCPRHY LCWSDVPVPR ISVIFSLEEL KEIEKDC AV YVGRMERVAR HSSVSKEEKE LRMEIAKQEL IAHAREAASR VLSALSDRQM SERMALDARK REQFQRLKEQ FVKDQERR Q AARQEELDDD FSYARELRDR ERRLKSLEEE LERKASKLSA EAARREQKAL WRIQRHRLES ARLRFLLEDE KHIQEMLKA VSEAHQPQEP PDVLLSVHPQ VTSPGPEHPE GGQGCDSGSA EQHSPAWDGW NRPGLLTPQP LKPLAVGAGG RGLQQAEGAR PFSDSLSIG DFLPVGPGAE PSVQTGMVPL LEVALQTINL DLPPSAPGEA PAAASTQPSR PQEYDFSTVL RPAVATSPAP G PLQAAECS LGSSGLQLWE DSCGKMDACG SASRETLLPS HPPRRAALEE GSSQPTERLF GQVSGGGLPT GDYASEIAPT RP RWNTHGH VSDASIRVGE NVSDVAPTQP RWNTHGHVSN ASISLGESVS DVAPTRPRWN IHGHVSNASI RVGENVSDVA PTR PRWNTH GHVSNASIRV GENVSDVAPT RPRWNTHGHV SDASISLGES VSDMAPARPR WNTHGHVSDA SISLGESVSD MAPT RPRWN THGHVSDTSI RVGENVSDVA PIRSRCNTHG HVSDASISLG EPVSDVVSTR PRWNTHVPIP PPHMVLGALS PEAEP NTPR PQQSPPGHTS QSALSLGAQS AVLDCGPRLP VEVGPSLSSP SSGCGEGSIS VGENVSDVAP TQPWWPNTPG DSVSEE LGP GRSGDTEDLS PNWPLNSQED TAAQSSPGRG EEAEASAAEA QGGEQAYLAG LAGQYHLERY PDSYESMSEP PIAHLLR PV LPRAFAFPVD PQVQSAADET AVQLSELLTL PVLMKRSITA PLAAHISLVN KAAVDYFFVE LHLEAHYEAL RHFLLMED G EFAQSLSDLL FEKLGAGQTP GELLNPLVLN SVLSKALQCS LHGDTPHASN LSLALKYLPE VFAPNAPDVL SCLELRYKV DWPLNIVITE GCLSKYSGVF SFLLQLKLMM WALKDVCFHL KRTALLSHMA GSVQFRQLQL FKHEMQHFVK VIQGYIANQI LHVTWCEFR ARLATVGDLE EIQRAHAEYL HEAVFRGLLT EKAAPVMNVI HSIFSLVLKF RSQLISQAWG PPGGPRGAEH P NFALMQQS YNTFKYYSHF LFKVVTKLVN RGYQPHLEDF LLRINFNNYY QDA

UniProtKB: TUBGCP6 protein

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Macromolecule #9: Gamma-tubulin complex component 5

MacromoleculeName: Gamma-tubulin complex component 5 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 118.467547 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MARHGPPWSR LDAQQERDVR ELVRGVAGLQ DEADPNFQLA LNFAWSNFRF HRFLDVNSHK IEKTIEGIYE KFVIHSDLSK AASWKRLTE EFLNAPLPSI KEIKTDAHYS ILSLLLCLSD SPSNSSYVET PRNKEVEKKD DFDWGKYLME DEEMDIGPYM D TPNWSEES ...String:
MARHGPPWSR LDAQQERDVR ELVRGVAGLQ DEADPNFQLA LNFAWSNFRF HRFLDVNSHK IEKTIEGIYE KFVIHSDLSK AASWKRLTE EFLNAPLPSI KEIKTDAHYS ILSLLLCLSD SPSNSSYVET PRNKEVEKKD DFDWGKYLME DEEMDIGPYM D TPNWSEES EEENDQQPLS REDSGIQVDR TPLEEQDQNR KLDPCISWKD EPDDRSWLEH HVVHQYWTAR PSQFPHSLHL HS NLAAVWD QHLYSSDPLY VPDDRVLVTE TQVIRETLWL LSGVKKLFIF QLIDGKVTVR NNIIVTHLTH SCLRSVLEQI AAY GQVVFR LQEFIDEVMG HSSESMLPGS GSVPKKSTEA PFRTYQAFMW ALYKYFISFK EELAEIEKCI INNDTTITLA IVVD KLAPR LSQLKVLHKV FSTGVAEVPP DTRNVVRASH LLNTLYKAIL EYDNVGEASE QTVSLLFSLW VETVRPYLQT VDEWI VHGH LWDGAREFII QRNKNVPVNH RDFWYATYTL YSVSEKTENE EKMSDNASAS SGSDQGPSSR QHTMVSFLKP VLKQII MAG KSMQLLKNLQ CAESTTCQAG ARDAERKSLY TLFLESVQSR LRHGEDSTPQ VLTEQQATKE NLMKMQSIAE SHLELDD VH DPLLAINFAR MYLEQSDFHE KFAGGDVCVD RSSESVTCQT FELTLRSCLY PHIDKQYLDC CGNLMQTLKK DYRLVEYL Q AMRNFFLMEG GDTMYDFYTS IFDKIREKET WQNVSFLNVQ LQEAVGQRYP EDSSRLSISF ENVDTAKKKL PVHILDGLT LSYKVPWPVD IVISLECQKI YNQVFLLLLQ IKWAKYSLDV LLFGELVSTA EKPRLKEGLI HEQDTVAQFG PQKEPVRQQI HRMFLLRVK LMHFVNSLHN YIMTRILHST GLEFQHQVEE AKDLDQLIKI HYRYLSTIHD RCLLREKVSF VKEAIMKVLN L ALMFADGW QAGLGTWRME SIEKMESDFK NCHMFLVTIL NKAVCRGSFP HLESLALSLM AGMEQS

UniProtKB: Gamma-tubulin complex component 5

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Macromolecule #10: Mitotic-spindle organizing protein 2B

MacromoleculeName: Mitotic-spindle organizing protein 2B / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.24665 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MAAQGVGPGP GSAAPPGLEA ARQKLALRRK KVLSTEEMEL YELAQAAGGA IDPDVFKILV DLLKLNVAPL AVFQMLKSMC AGQRLASEP QDPAAVSLPT SSVPETRGRN KGSAALGGAL ALAERSSREG SSQRMPRQPS ATRLPKGGGP GKSPTRGST

UniProtKB: Mitotic-spindle organizing protein 2B

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Macromolecule #11: CDK5 regulatory subunit-associated protein 2

MacromoleculeName: CDK5 regulatory subunit-associated protein 2 / type: protein_or_peptide / ID: 11 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 189.905844 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 EIRRREESFS LYSDQTSYLS ICLEENN RF QVEHFSQEEL KKKVSDLIQL VKELYTDNQH LKKTIFDLSC MGFQGNGFPD RLASTEQTEE AKKSRLPILI KPSRSLGN M YRLPATQEVV TQLQSQILEL QGELKEFKTC NKQLHQKLIL AEAVMEGRPT PDKTLLNAQP PVGAAYQDSP GEQKGIKTT SSVWRDKEMD SDQQRSYEID SEICPPDDLA SLPSCKENPE DVLSPTSVAT YLSSKSQPSA KVSVMGTDQS ESINTSNETE YLKQKIHDL ETELEGYQNF IFQLQKHSQC SEAIITVLCG TEGAQDGLSK PKNGSDGEEM TFSSLHQVRY VKHVKILGPL A PEMIDSRV LENLKQQLEE QEYKLQKEQN LNMQLFSEIH NLQNKFRDLS PPRYDSLVQS QARELSLQRQ QIKDGHGICV IS RQHMNTM IKAFEELLQA SDVDYCVAEG FQEQLNQCAE LLEKLEKLFL NGKSVGVEMN TQNELMERIE EDNLTYQHLL PES PEPSAS HALSDYETSE KSFFSRDQKQ DNETEKTSVM VNSFSQDLLM EHIQEIRTLR KRLEESIKTN EKLRKQLERQ GSEF VQGST SIFASGSELH SSLTSEIHFL RKQNQALNAM LIKGSRDKQK ENDKLRESLS RKTVSLEHLQ REYASVKEEN ERLQK EGSE KERHNQQLIQ EVRCSGQELS RVQEEVKLRQ QLLSQNDKLL QSLRVELKAY EKLDEEHRRL REASGEGWKG QDPFRD LHS LLMEIQALRL QLERSIETSS TLQSRLKEQL ARGAEKAQEG ALTLAVQAVS IPEVPLQPDK HDGDKYPMES DNSFDLF DS SQAVTPKSVS ETPPLSGNDT DSLSCDSGSS ATSTPCVSRL VTGHHLWASK NGRHVLGLIE DYEALLKQIS QGQRLLAE M DIQTQEAPSS TSQELGTKGP HPAPLSKFVS SVSTAKLTLE EAYRRLKLLW RVSLPEDGQC PLHCEQIGEM KAEVTKLHK KLFEQEKKLQ NTMKLLQLSK RQEKVIFDQL VVTHKILRKA RGNLELRPGG AHPGTCSPSR PGS

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
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS 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: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 71778
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9qvm:
Cryo-EM reconstruction of the NEDD1 anchor protein and CDK5RAP2 bound to the gamma-tubulin ring complex

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