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- EMDB-26347: Human CST bound to full-length DNA polymerase alpha-primase in a ... -

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Entry
Database: EMDB / ID: EMD-26347
TitleHuman CST bound to full-length DNA polymerase alpha-primase in a recruitment state
Map data
Sample
  • Complex: CST bound to full-length DNA polymerase alpha-primase and ssDNA
    • Protein or peptide: DNA primase catalytic subunit
    • Protein or peptide: DNA primase large subunit PRIM2
    • Protein or peptide: DNA polymerase alpha catalytic subunit POLA1
    • Protein or peptide: DNA polymerase alpha B subunit POLA2
    • Protein or peptide: CST complex subunit Ctc1
    • Protein or peptide: CST complex subunit Stn1
    • Protein or peptide: CST complex subunit Ten1
    • DNA: Telomeric G-strand ssDNA (3 repeats)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsCai SW / Zinder JC / Svetlov V / Bush MW / Nudler E / Walz T / de Lange T
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)5 R35 CA210036 United States
Breast Cancer Research FoundationBCRF-19-036 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Cryo-EM structure of the human CST-Polα/primase complex in a recruitment state.
Authors: Sarah W Cai / John C Zinder / Vladimir Svetlov / Martin W Bush / Evgeny Nudler / Thomas Walz / Titia de Lange /
Abstract: The CST-Polα/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of human CST- ...The CST-Polα/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of human CST-Polα/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus. Finally, Coats plus syndrome disease mutations previously characterized to disrupt formation of the CST-Polα/primase complex map to protein-protein interfaces observed in the recruitment state. Together, our results shed light on the architecture and stoichiometry of the metazoan fill-in machinery.
History
DepositionMar 2, 2022-
Header (metadata) releaseMay 18, 2022-
Map releaseMay 18, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26347.png

Downloads & links

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Map

FileDownload / File: emd_26347.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 100 pix.
= 300. Å		3 Å/pix.
x 100 pix.
= 300. Å		3 Å/pix.
x 100 pix.
= 300. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.049375683 - 0.08862725
Average (Standard dev.)0.0005345795 (±0.005753771)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 300.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_26347_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: #2

Fileemd_26347_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : CST bound to full-length DNA polymerase alpha-primase and ssDNA

EntireName: CST bound to full-length DNA polymerase alpha-primase and ssDNA
Components
  • Complex: CST bound to full-length DNA polymerase alpha-primase and ssDNA
    • Protein or peptide: DNA primase catalytic subunit
    • Protein or peptide: DNA primase large subunit PRIM2
    • Protein or peptide: DNA polymerase alpha catalytic subunit POLA1
    • Protein or peptide: DNA polymerase alpha B subunit POLA2
    • Protein or peptide: CST complex subunit Ctc1
    • Protein or peptide: CST complex subunit Stn1
    • Protein or peptide: CST complex subunit Ten1
    • DNA: Telomeric G-strand ssDNA (3 repeats)

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Supramolecule #1: CST bound to full-length DNA polymerase alpha-primase and ssDNA

SupramoleculeName: CST bound to full-length DNA polymerase alpha-primase and ssDNA
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all / Details: GraFix cross-linked
Molecular weightTheoretical: 540 KDa

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Macromolecule #1: DNA primase catalytic subunit

MacromoleculeName: DNA primase catalytic subunit / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: METFDPTELP ELLKLYYRRL FPYSQYYRWL NYGGVIKNYF QHREFSFTLK DDIYIRYQSF NNQSDLEKEM QKMNPYKIDI GAVYSHRPNQ HNTVKLGAFQ AQEKELVFDI DMTDYDDVRR CCSSADICPK CWTLMTMAIR IIDRALKEDF GFKHRLWVYS GRRGVHCWVC ...String:
METFDPTELP ELLKLYYRRL FPYSQYYRWL NYGGVIKNYF QHREFSFTLK DDIYIRYQSF NNQSDLEKEM QKMNPYKIDI GAVYSHRPNQ HNTVKLGAFQ AQEKELVFDI DMTDYDDVRR CCSSADICPK CWTLMTMAIR IIDRALKEDF GFKHRLWVYS GRRGVHCWVC DESVRKLSSA VRSGIVEYLS LVKGGQDVKK KVHLSEKIHP FIRKSINIIK KYFEEYALVN QDILENKESW DKILALVPET IHDELQQSFQ KSHNSLQRWE HLKKVASRYQ NNIKNDKYGP WLEWEIMLQY CFPRLDINVS KGINHLLKSP FSVHPKTGRI SVPIDLQKVD QFDPFTVPTI SFICRELDAI STNEEEKEEN EAESDVKHRT RDYKKTSLAP YVKVFEHFLE NLDKSRKGEL LKKSDLQKDF

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Macromolecule #2: DNA primase large subunit PRIM2

MacromoleculeName: DNA primase large subunit PRIM2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MEFSGRKWRK LRLAGDQRNA SYPHCLQFYL QPPSENISLI EFENLAIDRV KLLKSVENLG VSYVKGTEQY QSKLESELRK LKFSYRENLE DEYEPRRRDH ISHFILRLAY CQSEELRRWF IQQEMDLLRF RFSILPKDKI QDFLKDSQLQ FEAISDEEKT LREQEIVASS ...String:
MEFSGRKWRK LRLAGDQRNA SYPHCLQFYL QPPSENISLI EFENLAIDRV KLLKSVENLG VSYVKGTEQY QSKLESELRK LKFSYRENLE DEYEPRRRDH ISHFILRLAY CQSEELRRWF IQQEMDLLRF RFSILPKDKI QDFLKDSQLQ FEAISDEEKT LREQEIVASS PSLSGLKLGF ESIYKIPFAD ALDLFRGRKV YLEDGFAYVP LKDIVAIILN EFRAKLSKAL ALTARSLPAV QSDERLQPLL NHLSHSYTGQ DYSTQGNVGK ISLDQIDLLS TKSFPPCMRQ LHKALRENHH LRHGGRMQYG LFLKGIGLTL EQALQFWKQE FIKGKMDPDK FDKGYSYNIR HSFGKEGKRT DYTPFSCLKI ILSNPPSQGD YHGCPFRHSD PELLKQKLQS YKISPGGISQ ILDLVKGTHY QVACQKYFEM IHNVDDCGFS LNHPNQFFCE SQRILNGGKD IKKEPIQPET PQPKPSVQKT KDASSALASL NSSLEMDMEG LEDYFSEDS

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Macromolecule #3: DNA polymerase alpha catalytic subunit POLA1

MacromoleculeName: DNA polymerase alpha catalytic subunit POLA1 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAPVHGDDSL SDSGSFVSSR ARREKKSKKG RQEALERLKK AKAGEKYKYE VEDFTGVYEE VDEEQYSKLV QARQDDDWIV DDDGIGYVED GREIFDDDLE DDALDADEKG KDGKARNKDK RNVKKLAVTK PNNIKSMFIA CAGKKTADKA VDLSKDGLLG DILQDLNTET ...String:
MAPVHGDDSL SDSGSFVSSR ARREKKSKKG RQEALERLKK AKAGEKYKYE VEDFTGVYEE VDEEQYSKLV QARQDDDWIV DDDGIGYVED GREIFDDDLE DDALDADEKG KDGKARNKDK RNVKKLAVTK PNNIKSMFIA CAGKKTADKA VDLSKDGLLG DILQDLNTET PQITPPPVMI LKKKRSIGAS PNPFSVHTAT AVPSGKIASP VSRKEPPLTP VPLKRAEFAG DDVQVESTEE EQESGAMEFE DGDFDEPMEV EEVDLEPMAA KAWDKESEPA EEVKQEADSG KGTVSYLGSF LPDVSCWDID QEGDSSFSVQ EVQVDSSHLP LVKGADEEQV FHFYWLDAYE DQYNQPGVVF LFGKVWIESA ETHVSCCVMV KNIERTLYFL PREMKIDLNT GKETGTPISM KDVYEEFDEK IATKYKIMKF KSKPVEKNYA FEIPDVPEKS EYLEVKYSAE MPQLPQDLKG ETFSHVFGTN TSSLELFLMN RKIKGPCWLE VKSPQLLNQP VSWCKVEAMA LKPDLVNVIK DVSPPPLVVM AFSMKTMQNA KNHQNEIIAM AALVHHSFAL DKAAPKPPFQ SHFCVVSKPK DCIFPYAFKE VIEKKNVKVE VAATERTLLG FFLAKVHKID PDIIVGHNIY GFELEVLLQR INVCKAPHWS KIGRLKRSNM PKLGGRSGFG ERNATCGRMI CDVEISAKEL IRCKSYHLSE LVQQILKTER VVIPMENIQN MYSESSQLLY LLEHTWKDAK FILQIMCELN VLPLALQITN IAGNIMSRTL MGGRSERNEF LLLHAFYENN YIVPDKQIFR KPQQKLGDED EEIDGDTNKY KKGRKKAAYA GGLVLDPKVG FYDKFILLLD FNSLYPSIIQ EFNICFTTVQ RVASEAQKVT EDGEQEQIPE LPDPSLEMGI LPREIRKLVE RRKQVKQLMK QQDLNPDLIL QYDIRQKALK LTANSMYGCL GFSYSRFYAK PLAALVTYKG REILMHTKEM VQKMNLEVIY GDTDSIMINT NSTNLEEVFK LGNKVKSEVN KLYKLLEIDI DGVFKSLLLL KKKKYAALVV EPTSDGNYVT KQELKGLDIV RRDWCDLAKD TGNFVIGQIL SDQSRDTIVE NIQKRLIEIG ENVLNGSVPV SQFEINKALT KDPQDYPDKK SLPHVHVALW INSQGGRKVK AGDTVSYVIC QDGSNLTASQ RAYAPEQLQK QDNLTIDTQY YLAQQIHPVV ARICEPIDGI DAVLIATWLG LDPTQFRVHH YHKDEENDAL LGGPAQLTDE EKYRDCERFK CPCPTCGTEN IYDNVFDGSG TDMEPSLYRC SNIDCKASPL TFTVQLSNKL IMDIRRFIKK YYDGWLICEE PTCRNRTRHL PLQFSRTGPL CPACMKATLQ PEYSDKSLYT QLCFYRYIFD AECALEKLTT DHEKDKLKKQ FFTPKVLQDY RKLKNTAEQF LSRSGYSEVN LSKLFAGCAV KS

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Macromolecule #4: DNA polymerase alpha B subunit POLA2

MacromoleculeName: DNA polymerase alpha B subunit POLA2 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSASAQQLAE ELQIFGLDCE EALIEKLVEL CVQYGQNEEG MVGELIAFCT STHKVGLTSE ILNSFEHEFL SKRLSKARHS TCKDSGHAGA RDIVSIQELI EVEEEEEILL NSYTTPSKGS QKRAISTPET PLTKRSVSTR SPHQLLSPSS FSPSATPSQK YNSRSNRGEV ...String:
MSASAQQLAE ELQIFGLDCE EALIEKLVEL CVQYGQNEEG MVGELIAFCT STHKVGLTSE ILNSFEHEFL SKRLSKARHS TCKDSGHAGA RDIVSIQELI EVEEEEEILL NSYTTPSKGS QKRAISTPET PLTKRSVSTR SPHQLLSPSS FSPSATPSQK YNSRSNRGEV VTSFGLAQGV SWSGRGGAGN ISLKVLGCPE ALTGSYKSMF QKLPDIREVL TCKIEELGSE LKEHYKIEAF TPLLAPAQEP VTLLGQIGCD SNGKLNNKSV ILEGDREHSS GAQIPVDLSE LKEYSLFPGQ VVIMEGINTT GRKLVATKLY EGVPLPFYQP TEEDADFEQS MVLVACGPYT TSDSITYDPL LDLIAVINHD RPDVCILFGP FLDAKHEQVE NCLLTSPFED IFKQCLRTII EGTRSSGSHL VFVPSLRDVH HEPVYPQPPF SYSDLSREDK KQVQFVSEPC SLSINGVIFG LTSTDLLFHL GAEEISSSSG TSDRFSRILK HILTQRSYYP LYPPQEDMAI DYESFYVYAQ LPVTPDVLII PSELRYFVKD VLGCVCVNPG RLTKGQVGGT FARLYLRRPA ADGAERQSPC IAVQVVRI

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Macromolecule #5: CST complex subunit Ctc1

MacromoleculeName: CST complex subunit Ctc1 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAAGRAQVPS SEQAWLEDAQ VFIQKTLCPA VKEPNVQLTP LVIDCVKTVW LSQGRNQGST LPLSYSFVSV QDLKTHQRLP CCSHLSWSSS AYQAWAQEAG PNGNPLPREQ LLLLGTLTDL SADLEQECRN GSLYVRDNTG VLSCELIDLD LSWLGHLFLF PRWSYLPPAR ...String:
MAAGRAQVPS SEQAWLEDAQ VFIQKTLCPA VKEPNVQLTP LVIDCVKTVW LSQGRNQGST LPLSYSFVSV QDLKTHQRLP CCSHLSWSSS AYQAWAQEAG PNGNPLPREQ LLLLGTLTDL SADLEQECRN GSLYVRDNTG VLSCELIDLD LSWLGHLFLF PRWSYLPPAR WNSSGEGHLE LWDAPVPVFP LTISPGPVTP IPVLYPESAS CLLRLRNKLR GVQRNLAGSL VRLSALVKSK QKAYFILSLG RSHPAVTHVS IIVQVPAQLV WHRALRPGTA YVLTELRVSK IRGQRQHVWM TSQSSRLLLL KPECVQELEL ELEGPLLEAD PKPLPMPSNS EDKKDPESLV RYSRLLSYSG AVTGVLNEPA GLYELDGQLG LCLAYQQFRG LRRVMRPGVC LQLQDVHLLQ SVGGGTRRPV LAPCLRGAVL LQSFSRQKPG AHSSRQAYGA SLYEQLVWER QLGLPLYLWA TKALEELACK LCPHVLRHHQ FLQHSSPGSP SLGLQLLAPT LDLLAPPGSP VRNAHNEILE EPHHCPLQKY TRLQTPSSFP TLATLKEEGQ RKAWASFDPK ALLPLPEASY LPSCQLNRRL AWSWLCLLPS AFCPAQVLLG VLVASSHKGC LQLRDQSGSL PCLLLAKHSQ PLSDPRLIGC LVRAERFQLI VERDVRSSFP SWKELSMPGF IQKQQARVYV QFFLADALIL PVPRPCLHSA TPSTPQTDPT GPEGPHLGQS RLFLLCHKEA LMKRNFCVPP GASPEVPKPA LSFYVLGSWL GGTQRKEGTG WGLPEPQGND DNDQKVHLIF FGSSVRWFEF LHPGQVYRLI APGPATPMLF EKDGSSCISR RPLELAGCAS CLTVQDNWTL ELESSQDIQD VLDANKSLPE SSLTDLLSDN FTDSLVSFSA EILSRTLCEP LVASLWMKLG NTGAMRRCVK LTVALETAEC EFPPHLDVYI EDPHLPPSLG LLPGARVHFS QLEKRVSRSH NVYCCFRSST YVQVLSFPPE TTISIPLPHI YLAELLQGGQ SPFQATASCH IVSVFSLQLF WVCAYCTSIC RQGKCTRLGS TCPTQTAISQ AIIRLLVEDG TAEAVVTCRN HHVAAALGLC PREWASLLDF VQVPGRVVLQ FAGPGAQLES SARVDEPMTM FLWTLCTSPS VLRPIVLSFE LERKPSKIVP LEPPRLQRFQ CGELPFLTHV NPRLRLSCLS IRESEYSSSL GILASSC

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Macromolecule #6: CST complex subunit Stn1

MacromoleculeName: CST complex subunit Stn1 / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MQPGSSRCEE ETPSLLWGLD PVFLAFAKLY IRDILDMKES RQVPGVFLYN GHPIKQVDVL GTVIGVRERD AFYSYGVDDS TGVINCICWK KLNTESVSAA PSAARELSLT SQLKKLQETI EQKTKIEIGD TIRVRGSIRT YREEREIHAT TYYKVDDPVW NIQIARMLEL ...String:
MQPGSSRCEE ETPSLLWGLD PVFLAFAKLY IRDILDMKES RQVPGVFLYN GHPIKQVDVL GTVIGVRERD AFYSYGVDDS TGVINCICWK KLNTESVSAA PSAARELSLT SQLKKLQETI EQKTKIEIGD TIRVRGSIRT YREEREIHAT TYYKVDDPVW NIQIARMLEL PTIYRKVYDQ PFHSSALEKE EALSNPGALD LPSLTSLLSE KAKEFLMENR VQSFYQQELE MVESLLSLAN QPVIHSASSD QVNFKKDTTS KAIHSIFKNA IQLLQEKGLV FQKDDGFDNL YYVTREDKDL HRKIHRIIQQ DCQKPNHMEK GCHFLHILAC ARLSIRPGLS EAVLQQVLEL LEDQSDIVST MEHYYTAF

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Macromolecule #7: CST complex subunit Ten1

MacromoleculeName: CST complex subunit Ten1 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MMLPKPGTYY LPWEVSAGQV PDGSTLRTFG RLCLYDMIQS RVTLMAQHGS DQHQVLVCTK LVEPFHAQVG SLYIVLGELQ HQQDRGSVVK ARVLTCVEGM NLPLLEQAIR EQRLYKQERG GSQ

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Macromolecule #8: Telomeric G-strand ssDNA (3 repeats)

MacromoleculeName: Telomeric G-strand ssDNA (3 repeats) / type: dna / ID: 8 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
(DG)(DG)(DT)(DT)(DA)(DG)(DG)(DG)(DT)(DT) (DA)(DG)(DG)(DG)(DT)(DT)(DA)(DG)

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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
VitrificationCryogen name: ETHANE

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Image recordingImage recording ID: 1 / Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 57.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #1~

Microscopy ID1
MicroscopeFEI TALOS ARCTICA
Image recordingImage recording ID: 2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Image recording ID1
Particle selectionNumber selected: 748693
Final reconstructionResolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 109224
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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