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- EMDB-27107: Human CST-DNA polymerase alpha/primase preinitiation complex boun... -

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Basic information

Entry
Database: EMDB / ID: EMD-27107
TitleHuman CST-DNA polymerase alpha/primase preinitiation complex bound to 4xTEL-foldback template - PRIM2C advanced PIC
Map dataSharpened map
Sample
  • Complex: Human CST-DNA polymerase alpha/primase advanced preinitiation complex bound to 4xTEL-foldback DNA template
    • Protein or peptide: CST complex subunit CTC1
    • Protein or peptide: CST complex subunit STN1
    • Protein or peptide: CST complex subunit TEN1
    • Protein or peptide: DNA primase small subunit
    • Protein or peptide: DNA primase large subunit
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Protein or peptide: DNA polymerase alpha subunit B
    • DNA: DNA (5'-D(P*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*A)-3')
Keywordstelomere / C-strand / complex / 4xTEL-foldback DNA template / PRIM2C / REPLICATION-DNA complex
Function / homology
Function and homology information


CST complex / positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / telomerase inhibitor activity / DNA replication initiation / telomere maintenance via telomere lengthening / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 ...CST complex / positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / telomerase inhibitor activity / DNA replication initiation / telomere maintenance via telomere lengthening / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / Polymerase switching / telomere capping / alpha DNA polymerase:primase complex / Processive synthesis on the lagging strand / single-stranded telomeric DNA binding / DNA primase activity / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / primosome complex / G-rich strand telomeric DNA binding / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / bone marrow development / DNA strand elongation involved in DNA replication / intermediate filament cytoskeleton / hematopoietic stem cell proliferation / DNA synthesis involved in DNA repair / telomeric DNA binding / leading strand elongation / G1/S-Specific Transcription / DNA replication origin binding / negative regulation of telomere maintenance via telomerase / replicative senescence / DNA replication initiation / Activation of the pre-replicative complex / spleen development / regulation of G2/M transition of mitotic cell cycle / telomere maintenance / thymus development / positive regulation of DNA replication / Defective pyroptosis / multicellular organism growth / fibrillar center / double-strand break repair via nonhomologous end joining / nuclear matrix / protein import into nucleus / positive regulation of fibroblast proliferation / nuclear envelope / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / chromosome, telomeric region / DNA-directed DNA polymerase activity / intracellular membrane-bounded organelle / DNA repair / nucleotide binding / DNA damage response / chromatin binding / chromatin / nucleolus / protein kinase binding / magnesium ion binding / DNA binding / zinc ion binding / nucleoplasm / membrane / nucleus / metal ion binding / cytosol
Similarity search - Function
CST complex subunit Ten1, animal and plant type / CST complex subunit CTC1 / CST complex subunit CTC1-like / CST, telomere maintenance, complex subunit CTC1 / Telomere-capping, CST complex subunit / CST complex subunit Stn1 / Stn1, C-terminal / CST complex subunit Stn1, wHTH1 motif superfamily / CST, complex subunit STN1, C terminal / Replication factor A protein-like ...CST complex subunit Ten1, animal and plant type / CST complex subunit CTC1 / CST complex subunit CTC1-like / CST, telomere maintenance, complex subunit CTC1 / Telomere-capping, CST complex subunit / CST complex subunit Stn1 / Stn1, C-terminal / CST complex subunit Stn1, wHTH1 motif superfamily / CST, complex subunit STN1, C terminal / Replication factor A protein-like / DNA polymerase alpha, subunit B, N-terminal domain superfamily / : / DNA polymerase alpha subunit B, OB domain / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase alpha catalytic subunit / DNA primase small subunit / DNA primase large subunit / DNA polymerase alpha subunit B / CST complex subunit CTC1 / CST complex subunit TEN1 / CST complex subunit STN1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.27 Å
AuthorsHe Q / Lin X / Chavez BL / Agrawal S / Lusk BL / Lim C
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM131023 United States
CitationJournal: Nature / Year: 2022
Title: Structures of the human CST-Polα-primase complex bound to telomere templates.
Authors: Qixiang He / Xiuhua Lin / Bianca L Chavez / Sourav Agrawal / Benjamin L Lusk / Ci Ji Lim /
Abstract: The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways such as lagging-strand ...The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.
History
DepositionMay 26, 2022-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27107.png

Downloads & links

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Map

FileDownload / File: emd_27107.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 400 pix.
= 440. Å		1.1 Å/pix.
x 400 pix.
= 440. Å		1.1 Å/pix.
x 400 pix.
= 440. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.81352377 - 1.7242358
Average (Standard dev.)0.001783855 (±0.040166818)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 440.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_27107_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Half map: Half Map 1

Fileemd_27107_half_map_1.map
AnnotationHalf Map 1
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Half map: Half Map 2

Fileemd_27107_half_map_2.map
AnnotationHalf Map 2
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Sample components

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Entire : Human CST-DNA polymerase alpha/primase advanced preinitiation com...

EntireName: Human CST-DNA polymerase alpha/primase advanced preinitiation complex bound to 4xTEL-foldback DNA template
Components
  • Complex: Human CST-DNA polymerase alpha/primase advanced preinitiation complex bound to 4xTEL-foldback DNA template
    • Protein or peptide: CST complex subunit CTC1
    • Protein or peptide: CST complex subunit STN1
    • Protein or peptide: CST complex subunit TEN1
    • Protein or peptide: DNA primase small subunit
    • Protein or peptide: DNA primase large subunit
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Protein or peptide: DNA polymerase alpha subunit B
    • DNA: DNA (5'-D(P*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*A)-3')

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Supramolecule #1: Human CST-DNA polymerase alpha/primase advanced preinitiation com...

SupramoleculeName: Human CST-DNA polymerase alpha/primase advanced preinitiation complex bound to 4xTEL-foldback DNA template
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fold-back double-stranded DNA region of the DNA template is not modeled due to structural flexibility.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 540 KDa

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Macromolecule #1: CST complex subunit CTC1

MacromoleculeName: CST complex subunit CTC1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 138.03025 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGDYKDHDGD YKDHDIDYKD DDDKSGGSLE AAGRAQVPSS EQAWLEDAQV FIQKTLCPAV KEPNVQLTPL VIDCVKTVWL SQGRNQGST LPLSYSFVSV QDLKTHQRLP CCSHLSWSSS AYQAWAQEAG PNGNPLPREQ LLLLGTLTDL SADLEQECRN G SLYVRDNT ...String:
MGDYKDHDGD YKDHDIDYKD DDDKSGGSLE AAGRAQVPSS EQAWLEDAQV FIQKTLCPAV KEPNVQLTPL VIDCVKTVWL SQGRNQGST LPLSYSFVSV QDLKTHQRLP CCSHLSWSSS AYQAWAQEAG PNGNPLPREQ LLLLGTLTDL SADLEQECRN G SLYVRDNT GVLSCELIDL DLSWLGHLFL FPRWSYLPPA RWNSSGEGHL ELWDAPVPVF PLTISPGPVT PIPVLYPESA SC LLRLRNK LRGVQRNLAG SLVRLSALVK SKQKAYFILS LGRSHPAVTH VSIIVQVPAQ LVWHRALRPG TAYVLTELRV SKI RGQRQH VWMTSQSSRL LLLKPECVQE LELELEGPLL EADPKPLPMP SNSEDKKDPE SLVRYSRLLS YSGAVTGVLN EPAG LYELD GQLGLCLAYQ QFRGLRRVMR PGVCLQLQDV HLLQSVGGGT RRPVLAPCLR GAVLLQSFSR QKPGAHSSRQ AYGAS LYEQ LVWERQLGLP LYLWATKALE ELACKLCPHV LRHHQFLQHS SPGSPSLGLQ LLAPTLDLLA PPGSPVRNAH NEILEE PHH CPLQKYTRLQ TPSSFPTLAT LKEEGQRKAW ASFDPKALLP LPEASYLPSC QLNRRLAWSW LCLLPSAFCP AQVLLGV LV ASSHKGCLQL RDQSGSLPCL LLAKHSQPLS DPRLIGCLVR AERFQLIVER DVRSSFPSWK ELSMPGFIQK QQARVYVQ F FLADALILPV PRPCLHSATP STPQTDPTGP EGPHLGQSRL FLLCHKEALM KRNFCVPPGA SPEVPKPALS FYVLGSWLG GTQRKEGTGW GLPEPQGNDD NDQKVHLIFF GSSVRWFEFL HPGQVYRLVA PGPATPMLFE KDGSSCISRR PLELAGCASC LTVQDNWTL ELESSQDIQD VLDANKSLPE SSLTDLLSDN FTDSLVSFSA EILSRTLCEP LVASLWMKLG NTGAMRRCVK L TVALETAE CEFPPHLDVY IEDPHLPPSL GLLPGARVHF SQLEKRVSRS HNVYCCFRSS TYVQVLSFPP ETTISVPLPH IY LAELLQG GQSPFQATAS CHIVSVFSLQ LFWVCAYCTS ICRQGKCTRL GSTCPTQTAI SQAIIRLLVE DGTAEAVVTC RNH HVAAAL GLCPREWASL LDFVQVPGRV VLQFAGPGAQ LESSARVDEP MTMFLWTLCT SPSVLRPIVL SFELERKPSK IVPL EPPRL QRFQCGELPF LTHVNPRLRL SCLSIRESEY SSSLGILASS C

UniProtKB: CST complex subunit CTC1

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Macromolecule #2: CST complex subunit STN1

MacromoleculeName: CST complex subunit STN1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.001824 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MHHHHHHQPG SSRCEEETPS LLWGLDPVFL AFAKLYIRDI LDMKESRQVP GVFLYNGHPI KQVDVLGTVI GVRERDAFYS YGVDDSTGV INCICWKKLN TESVSAAPSA ARELSLTSQL KKLQETIEQK TKIEIGDTIR VRGSIRTYRE EREIHATTYY K VDDPVWNI ...String:
MHHHHHHQPG SSRCEEETPS LLWGLDPVFL AFAKLYIRDI LDMKESRQVP GVFLYNGHPI KQVDVLGTVI GVRERDAFYS YGVDDSTGV INCICWKKLN TESVSAAPSA ARELSLTSQL KKLQETIEQK TKIEIGDTIR VRGSIRTYRE EREIHATTYY K VDDPVWNI QIARMLELPT IYRKVYDQPF HSSALEKEEA LSNPGALDLP SLTSLLSEKA KEFLMENRVQ SFYQQELEMV ES LLSLANQ PVIHSASSDQ VNFKKDTTSK AIHSIFKNAI QLLQEKGLVF QKDDGFDNLY YVTREDKDLH RKIHRIIQQD CQK PNHMEK GCHFLHILAC ARLSIRPGLS EAVLQQVLEL LEDQSDIVST MEHYYTAF

UniProtKB: CST complex subunit STN1

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Macromolecule #3: CST complex subunit TEN1

MacromoleculeName: CST complex subunit TEN1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.285604 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MSYYHHHHHH DYDIPTTENL YFQGAMGSGI QLPKPGTYYL PWEVSAGQVP DGSTLRTFGR LCLYDMIQSR VTLMAQHGSD QHQVLVCTK LVEPFHAQVG SLYIVLGELQ HQQDRGSVVK ARVLTCVEGM NLPLLEQAIR EQRLYKQERG GSQ

UniProtKB: CST complex subunit TEN1

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Macromolecule #4: DNA primase small subunit

MacromoleculeName: DNA primase small subunit / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA primase AEP
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.356379 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHGS GSGSGETFDP TELPELLKLY YRRLFPYSQY YRWLNYGGVI KNYFQHREFS FTLKDDIYIR YQSFNNQSDL EKEMQKMNP YKIDIGAVYS HRPNQHNTVK LGAFQAQEKE LVFDIDMTDY DDVRRCCSSA DICPKCWTLM TMAIRIIDRA L KEDFGFKH ...String:
MGHHHHHHGS GSGSGETFDP TELPELLKLY YRRLFPYSQY YRWLNYGGVI KNYFQHREFS FTLKDDIYIR YQSFNNQSDL EKEMQKMNP YKIDIGAVYS HRPNQHNTVK LGAFQAQEKE LVFDIDMTDY DDVRRCCSSA DICPKCWTLM TMAIRIIDRA L KEDFGFKH RLWVYSGRRG VHCWVCDESV RKLSSAVRSG IVEYLSLVKG GQDVKKKVHL SEKIHPFIRK SINIIKKYFE EY ALVNQDI LENKESWDKI LALVPETIHD ELQQSFQKSH NSLQRWEHLK KVASRYQNNI KNDKYGPWLE WEIMLQYCFP RLD INVSKG INHLLKSPFS VHPKTGRISV PIDLQKVDQF DPFTVPTISF ICRELDAIST NEEEKEENEA ESDVKHRTRD YKKT SLAPY VKVFEHFLEN LDKSRKGELL KKSDLQKDF

UniProtKB: DNA primase small subunit

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Macromolecule #5: DNA primase large subunit

MacromoleculeName: DNA primase large subunit / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60.266293 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHGS GSGSGEFSGR KWRKLRLAGD QRNASYPHCL QFYLQPPSEN ISLIEFENLA IDRVKLLKSV ENLGVSYVKG TEQYQSKLE SELRKLKFSY RENLEDEYEP RRRDHISHFI LRLAYCQSEE LRRWFIQQEM DLLRFRFSIL PKDKIQDFLK D SQLQFEAI ...String:
MGHHHHHHGS GSGSGEFSGR KWRKLRLAGD QRNASYPHCL QFYLQPPSEN ISLIEFENLA IDRVKLLKSV ENLGVSYVKG TEQYQSKLE SELRKLKFSY RENLEDEYEP RRRDHISHFI LRLAYCQSEE LRRWFIQQEM DLLRFRFSIL PKDKIQDFLK D SQLQFEAI SDEEKTLREQ EIVASSPSLS GLKLGFESIY KIPFADALDL FRGRKVYLED GFAYVPLKDI VAIILNEFRA KL SKALALT ARSLPAVQSD ERLQPLLNHL SHSYTGQDYS TQGNVGKISL DQIDLLSTKS FPPCMRQLHK ALRENHHLRH GGR MQYGLF LKGIGLTLEQ ALQFWKQEFI KGKMDPDKFD KGYSYNIRHS FGKEGKRTDY TPFSCLKIIL SNPPSQGDYH GCPF RHSDP ELLKQKLQSY KISPGGISQI LDLVKGTHYQ VACQKYFEMI HNVDDCGFSL NHPNQFFCES QRILNGGKDI KKEPI QPET PQPKPSVQKT KDASSALASL NSSLEMDMEG LEDYFSEDS

UniProtKB: DNA primase large subunit

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Macromolecule #6: DNA polymerase alpha catalytic subunit

MacromoleculeName: DNA polymerase alpha catalytic subunit / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 172.796859 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGGSAGDYKD HDGDYKDHDI DYKDDDDKGA SSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KGAGSAPVHG DDSLSDSGSF VSSRARREK KSKKGRQEAL ERLKKAKAGE KYKYEVEDFT GVYEEVDEEQ YSKLVQARQD DDWIVDDDGI GYVEDGREIF D DDLEDDAL ...String:
MGGSAGDYKD HDGDYKDHDI DYKDDDDKGA SSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KGAGSAPVHG DDSLSDSGSF VSSRARREK KSKKGRQEAL ERLKKAKAGE KYKYEVEDFT GVYEEVDEEQ YSKLVQARQD DDWIVDDDGI GYVEDGREIF D DDLEDDAL DADEKGKDGK ARNKDKRNVK KLAVTKPNNI KSMFIACAGK KTADKAVDLS KDGLLGDILQ DLNTETPQIT PP PVMILKK KRSIGASPNP FSVHTATAVP SGKIASPVSR KEPPLTPVPL KRAEFAGDDV QVESTEEEQE SGAMEFEDGD FDE PMEVEE VDLEPMAAKA WDKESEPAEE VKQEADSGKG TVSYLGSFLP DVSCWDIDQE GDSSFSVQEV QVDSSHLPLV KGAD EEQVF HFYWLDAYED QYNQPGVVFL FGKVWIESAE THVSCCVMVK NIERTLYFLP REMKIDLNTG KETGTPISMK DVYEE FDEK IATKYKIMKF KSKPVEKNYA FEIPDVPEKS EYLEVKYSAE MPQLPQDLKG ETFSHVFGTN TSSLELFLMN RKIKGP CWL EVKSPQLLNQ PVSWCKVEAM ALKPDLVNVI KDVSPPPLVV MAFSMKTMQN AKNHQNEIIA MAALVHHSFA LDKAAPK PP FQSHFCVVSK PKDCIFPYAF KEVIEKKNVK VEVAATERTL LGFFLAKVHK IDPDIIVGHN IYGFELEVLL QRINVCKA P HWSKIGRLKR SNMPKLGGRS GFGERNATCG RMICDVEISA KELIRCKSYH LSELVQQILK TERVVIPMEN IQNMYSESS QLLYLLEHTW KDAKFILQIM CELNVLPLAL QITNIAGNIM SRTLMGGRSE RNEFLLLHAF YENNYIVPDK QIFRKPQQKL GDEDEEIDG DTNKYKKGRK KAAYAGGLVL DPKVGFYDKF ILLLDFNSLY PSIIQEFNIC FTTVQRVASE AQKVTEDGEQ E QIPELPDP SLEMGILPRE IRKLVERRKQ VKQLMKQQDL NPDLILQYDI RQKALKLTAN SMYGCLGFSY SRFYAKPLAA LV TYKGREI LMHTKEMVQK MNLEVIYGDT DSIMINTNST NLEEVFKLGN KVKSEVNKLY KLLEIDIDGV FKSLLLLKKK KYA ALVVEP TSDGNYVTKQ ELKGLDIVRR DWCDLAKDTG NFVIGQILSD QSRDTIVENI QKRLIEIGEN VLNGSVPVSQ FEIN KALTK DPQDYPDKKS LPHVHVALWI NSQGGRKVKA GDTVSYVICQ DGSNLTASQR AYAPEQLQKQ DNLTIDTQYY LAQQI HPVV ARICEPIDGI DAVLIATWLG LDPTQFRVHH YHKDEENDAL LGGPAQLTDE EKYRDCERFK CPCPTCGTEN IYDNVF DGS GTDMEPSLYR CSNIDCKASP LTFTVQLSNK LIMDIRRFIK KYYDGWLICE EPTCRNRTRH LPLQFSRTGP LCPACMK AT LQPEYSDKSL YTQLCFYRYI FDAECALEKL TTDHEKDKLK KQFFTPKVLQ DYRKLKNTAE QFLSRSGYSE VNLSKLFA G CAVKSV

UniProtKB: DNA polymerase alpha catalytic subunit

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Macromolecule #7: DNA polymerase alpha subunit B

MacromoleculeName: DNA polymerase alpha subunit B / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 67.390898 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHGS GSGSGSASAQ QLAEELQIFG LDCEEALIEK LVELCVQYGQ NEEGMVGELI AFCTSTHKVG LTSEILNSFE HEFLSKRLS KARHSTCKDS GHAGARDIVS IQELIEVEEE EEILLNSYTT PSKGSQKRAI STPETPLTKR SVSTRSPHQL L SPSSFSPS ...String:
MGHHHHHHGS GSGSGSASAQ QLAEELQIFG LDCEEALIEK LVELCVQYGQ NEEGMVGELI AFCTSTHKVG LTSEILNSFE HEFLSKRLS KARHSTCKDS GHAGARDIVS IQELIEVEEE EEILLNSYTT PSKGSQKRAI STPETPLTKR SVSTRSPHQL L SPSSFSPS ATPSQKYNSR SNRGEVVTSF GLAQGVSWSG RGGAGNISLK VLGCPEALTG SYKSMFQKLP DIREVLTCKI EE LGSELKE HYKIEAFTPL LAPAQEPVTL LGQIGCDSNG KLNNKSVILE GDREHSSGAQ IPVDLSELKE YSLFPGQVVI MEG INTTGR KLVATKLYEG VPLPFYQPTE EDADFEQSMV LVACGPYTTS DSITYDPLLD LIAVINHDRP DVCILFGPFL DAKH EQVEN CLLTSPFEDI FKQCLRTIIE GTRSSGSHLV FVPSLRDVHH EPVYPQPPFS YSDLSREDKK QVQFVSEPCS LSING VIFG LTSTDLLFHL GAEEISSSSG TSDRFSRILK HILTQRSYYP LYPPQEDMAI DYESFYVYAQ LPVTPDVLII PSELRY FVK DVLGCVCVNP GRLTKGQVGG TFARLYLRRP AADGAERQSP CIAVQVVRI

UniProtKB: DNA polymerase alpha subunit B

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Macromolecule #8: DNA (5'-D(P*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP...

MacromoleculeName: DNA (5'-D(P*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*A)-3')
type: dna / ID: 8
Details: Residue 1-35 forms a double-stranded DNA hairpin stem at the 5' end of the single-stranded DNA telomeric template.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.67092 KDa
SequenceString: (DC)(DT)(DA)(DA)(DC)(DC)(DG)(DC)(DA)(DT) (DC)(DT)(DA)(DG)(DC)(DT)(DT)(DT)(DT)(DT) (DG)(DC)(DT)(DA)(DG)(DA)(DT)(DG)(DC) (DG)(DG)(DT)(DT)(DA)(DG)(DC)(DT)(DT)(DA) (DG) (DG)(DG)(DT)(DT)(DA)(DG) ...String:
(DC)(DT)(DA)(DA)(DC)(DC)(DG)(DC)(DA)(DT) (DC)(DT)(DA)(DG)(DC)(DT)(DT)(DT)(DT)(DT) (DG)(DC)(DT)(DA)(DG)(DA)(DT)(DG)(DC) (DG)(DG)(DT)(DT)(DA)(DG)(DC)(DT)(DT)(DA) (DG) (DG)(DG)(DT)(DT)(DA)(DG)(DG)(DG) (DT)(DT)(DA)(DG)(DG)(DG)(DT)(DT)(DA)(DG) (DG)(DG)

GENBANK: GENBANK: LR877228.1

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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

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
50.0 mMHEPES-Na
150.0 mMNaCl
2.0 mMMgCl2
1.0 mMTCEP
4.0 mMCHAPSO

Details: CHAPSO is only added just before sample vitrification.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7794 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.7000000000000001 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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 32550
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsAlphafold models were used to dock into the map before coot adjustments and phenix refinement.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross coefficient
Output model

PDB-8d0k:
Human CST-DNA polymerase alpha/primase preinitiation complex bound to 4xTEL-foldback template - PRIM2C advanced PIC

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