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- EMDB-27104: Human CST-DNA polymerase alpha/primase preinitiation complex boun... -

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Basic information

Entry
Database: EMDB / ID: EMD-27104
TitleHuman CST-DNA polymerase alpha/primase preinitiation complex bound to 4xTEL-foldback template
Map dataMerged map using three separately refined domains
Sample
  • Complex: Human CST-DNA polymerase alpha/primase 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*G)-3')
Keywordstelomere / C-strand / complex / 4xTEL-foldback DNA template / 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: 3.43 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_27104.png

Downloads & links

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Map

FileDownload / File: emd_27104.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMerged map using three separately refined domains
Projections & slices

Image control

Size
Brightness
Contrast
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

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-0.13345061 - 2.8543658
Average (Standard dev.)0.02124728 (±0.05162174)
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: Consensus map

Fileemd_27104_additional_1.map
AnnotationConsensus map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus half map 2

Fileemd_27104_half_map_1.map
AnnotationConsensus half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus half map 1

Fileemd_27104_half_map_2.map
AnnotationConsensus half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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

EntireName: Human CST-DNA polymerase alpha/primase preinitiation complex bound to 4xTEL-foldback DNA template
Components
  • Complex: Human CST-DNA polymerase alpha/primase 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*G)-3')

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Supramolecule #1: Human CST-DNA polymerase alpha/primase preinitiation complex boun...

SupramoleculeName: Human CST-DNA polymerase alpha/primase 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 and PRIM2 C-term domain are 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: 133.938094 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: PSSEQAWLED AQVFIQKTLC PAVKEPNVQL TPLVIDCVKT VWLSQGRNQG STLPLSYSFV SVQDLKTHQR LPCCSHLSWS SSAYQAWAQ EAGPNGNPLP REQLLLLGTL TDLSADLEQE CRNGSLYVRD NTGVLSCELI DLDLSWLGHL FLFPRWSYLP P ARWNSSGE ...String:
PSSEQAWLED AQVFIQKTLC PAVKEPNVQL TPLVIDCVKT VWLSQGRNQG STLPLSYSFV SVQDLKTHQR LPCCSHLSWS SSAYQAWAQ EAGPNGNPLP REQLLLLGTL TDLSADLEQE CRNGSLYVRD NTGVLSCELI DLDLSWLGHL FLFPRWSYLP P ARWNSSGE GHLELWDAPV PVFPLTISPG PVTPIPVLYP ESASCLLRLR NKLRGVQRNL AGSLVRLSAL VKSKQKAYFI LS LGRSHPA VTHVSIIVQV PAQLVWHRAL RPGTAYVLTE LRVSKIRGQR QHVWMTSQSS RLLLLKPECV QELELELEGP LLE ADPKPL PMPSNSEDKK DPESLVRYSR LLSYSGAVTG VLNEPAGLYE LDGQLGLCLA YQQFRGLRRV MRPGVCLQLQ DVHL LQSVG GGTRRPVLAP CLRGAVLLQS FSRQKPGAHS SRQAYGASLY EQLVWERQLG LPLYLWATKA LEELACKLCP HVLRH HQFL QHSSPGSPSL GLQLLAPTLD LLAPPGSPVR NAHNEILEEP HHCPLQKYTR LQTPSSFPTL ATLKEEGQRK AWASFD PKA LLPLPEASYL PSCQLNRRLA WSWLCLLPSA FCPAQVLLGV LVASSHKGCL QLRDQSGSLP CLLLAKHSQP LSDPRLI GC LVRAERFQLI VERDVRSSFP SWKELSMPGF IQKQQARVYV QFFLADALIL PVPRPCLHSA TPSTPQTDPT GPEGPHLG Q SRLFLLCHKE ALMKRNFCVP PGASPEVPKP ALSFYVLGSW LGGTQRKEGT GWGLPEPQGN DDNDQKVHLI FFGSSVRWF EFLHPGQVYR LVAPGPATPM LFEKDGSSCI SRRPLELAGC ASCLTVQDNW TLELESSQDI QDVLDANKSL PESSLTDLLS DNFTDSLVS FSAEILSRTL CEPLVASLWM KLGNTGAMRR CVKLTVALET AECEFPPHLD VYIEDPHLPP SLGLLPGARV H FSQLEKRV SRSHNVYCCF RSSTYVQVLS FPPETTISVP LPHIYLAELL QGGQSPFQAT ASCHIVSVFS LQLFWVCAYC TS ICRQGKC TRLGSTCPTQ TAISQAIIRL LVEDGTAEAV VTCRNHHVAA ALGLCPREWA SLLDFVQVPG RVVLQFAGPG AQL ESSARV DEPMTMFLWT LCTSPSVLRP IVLSFELERK PSKIVPLEPP RLQRFQCGEL PFLTHVNPRL RLSCLSIRES EYSS SLGIL ASSC

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: 41.585305 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: RCEEETPSLL WGLDPVFLAF AKLYIRDILD MKESRQVPGV FLYNGHPIKQ VDVLGTVIGV RERDAFYSYG VDDSTGVINC ICWKKLNTE SVSAAPSAAR ELSLTSQLKK LQETIEQKTK IEIGDTIRVR GSIRTYREER EIHATTYYKV DDPVWNIQIA R MLELPTIY ...String:
RCEEETPSLL WGLDPVFLAF AKLYIRDILD MKESRQVPGV FLYNGHPIKQ VDVLGTVIGV RERDAFYSYG VDDSTGVINC ICWKKLNTE SVSAAPSAAR ELSLTSQLKK LQETIEQKTK IEIGDTIRVR GSIRTYREER EIHATTYYKV DDPVWNIQIA R MLELPTIY RKVYDQPFHS SALEKEEALS NPGALDLPSL TSLLSEKAKE FLMENRVQSF YQQELEMVES LLSLANQPVI HS ASSDQVN FKKDTTSKAI HSIFKNAIQL LQEKGLVFQK DDGFDNLYYV TREDKDLHRK IHRIIQQDCQ KPNHMEKGCH FLH ILACAR LSIRPGLSEA VLQQVLELLE DQSDIVSTME HYYTAF

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: 13.609621 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
LPKPGTYYLP WEVSAGQVPD GSTLRTFGRL CLYDMIQSRV TLMAQHGSDQ HQVLVCTKLV EPFHAQVGSL YIVLGELQHQ QDRGSVVKA RVLTCVEGMN LPLLEQAIRE QRLYKQERGG SQ

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: 49.849812 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: ETFDPTELPE LLKLYYRRLF PYSQYYRWLN YGGVIKNYFQ HREFSFTLKD DIYIRYQSFN NQSDLEKEMQ KMNPYKIDIG AVYSHRPNQ HNTVKLGAFQ AQEKELVFDI DMTDYDDVRR CCSSADICPK CWTLMTMAIR IIDRALKEDF GFKHRLWVYS G RRGVHCWV ...String:
ETFDPTELPE LLKLYYRRLF PYSQYYRWLN YGGVIKNYFQ HREFSFTLKD DIYIRYQSFN NQSDLEKEMQ KMNPYKIDIG AVYSHRPNQ HNTVKLGAFQ AQEKELVFDI DMTDYDDVRR CCSSADICPK CWTLMTMAIR IIDRALKEDF GFKHRLWVYS G RRGVHCWV CDESVRKLSS AVRSGIVEYL SLVKGGQDVK KKVHLSEKIH PFIRKSINII KKYFEEYALV NQDILENKES WD KILALVP ETIHDELQQS FQKSHNSLQR WEHLKKVASR YQNNIKNDKY GPWLEWEIML QYCFPRLDIN VSKGINHLLK SPF SVHPKT GRISVPIDLQ KVDQFDPFTV PTISFICREL DAISTNEEEK EENEAESDVK HRTRDYKKTS LAPYVKVFEH FLEN LDKSR KGELLKKSDL QKDF

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: 28.272275 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: DQRNASYPHC LQFYLQPPSE NISLIEFENL AIDRVKLLKS VENLGVSYVK GTEQYQSKLE SELRKLKFSY RENLEDEYEP RRRDHISHF ILRLAYCQSE ELRRWFIQQE MDLLRFRFSI LPKDKIQDFL KDSQLQFEAI SDEEKTLREQ EIVASSPSLS G LKLGFESI ...String:
DQRNASYPHC LQFYLQPPSE NISLIEFENL AIDRVKLLKS VENLGVSYVK GTEQYQSKLE SELRKLKFSY RENLEDEYEP RRRDHISHF ILRLAYCQSE ELRRWFIQQE MDLLRFRFSI LPKDKIQDFL KDSQLQFEAI SDEEKTLREQ EIVASSPSLS G LKLGFESI YKIPFADALD LFRGRKVYLE DGFAYVPLKD IVAIILNEFR AKLSKALALT ARSLPAVQSD ERLQPLLNHL SH S

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: 130.472156 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: VDSSHLPLVK GADEEQVFHF YWLDAYEDQY NQPGVVFLFG KVWIESAETH VSCCVMVKNI ERTLYFLPRE MKIDLNTGKE TGTPISMKD VYEEFDEKIA TKYKIMKFKS KPVEKNYAFE IPDVPEKSEY LEVKYSAEMP QLPQDLKGET FSHVFGTNTS S LELFLMNR ...String:
VDSSHLPLVK GADEEQVFHF YWLDAYEDQY NQPGVVFLFG KVWIESAETH VSCCVMVKNI ERTLYFLPRE MKIDLNTGKE TGTPISMKD VYEEFDEKIA TKYKIMKFKS KPVEKNYAFE IPDVPEKSEY LEVKYSAEMP QLPQDLKGET FSHVFGTNTS S LELFLMNR KIKGPCWLEV KSPQLLNQPV SWCKVEAMAL KPDLVNVIKD VSPPPLVVMA FSMKTMQNAK NHQNEIIAMA AL VHHSFAL DKAAPKPPFQ SHFCVVSKPK DCIFPYAFKE VIEKKNVKVE VAATERTLLG FFLAKVHKID PDIIVGHNIY GFE LEVLLQ RINVCKAPHW SKIGRLKRSN MPKLGGRSGF GERNATCGRM ICDVEISAKE LIRCKSYHLS ELVQQILKTE RVVI PMENI QNMYSESSQL LYLLEHTWKD AKFILQIMCE LNVLPLALQI TNIAGNIMSR TLMGGRSERN EFLLLHAFYE NNYIV PDKQ IFRKPQQKLG DEDEEIDGDT NKYKKGRKKA AYAGGLVLDP KVGFYDKFIL LLDFNSLYPS IIQEFNICFT TVQRVA SEA QKVTEDGEQE QIPELPDPSL EMGILPREIR KLVERRKQVK QLMKQQDLNP DLILQYDIRQ KALKLTANSM YGCLGFS YS RFYAKPLAAL VTYKGREILM HTKEMVQKMN LEVIYGDTDS IMINTNSTNL EEVFKLGNKV KSEVNKLYKL LEIDIDGV F KSLLLLKKKK YAALVVEPTS DGNYVTKQEL KGLDIVRRDW CDLAKDTGNF VIGQILSDQS RDTIVENIQK RLIEIGENV LNGSVPVSQF EINKALTKDP QDYPDKKSLP HVHVALWINS QGGRKVKAGD TVSYVICQDG SNLTASQRAY APEQLQKQDN LTIDTQYYL AQQIHPVVAR ICEPIDGIDA VLIATWLGLD PTQFRVHHYH KDEENDALLG GPAQLTDEEK YRDCERFKCP C PTCGTENI YDNVFDGSGT DMEPSLYRCS NIDCKASPLT FTVQLSNKLI MDIRRFIKKY YDGWLICEEP TCRNRTRHLP LQ FSRTGPL CPACMKATLQ PEYSDKSLYT QLCFYRYIFD AECALEKLTT DHEKDKLKKQ FFTPKVLQDY RKLKNTAEQF LSR SGYSEV NLSKLFAGCA VKS

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: 50.343969 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: HQLLSPSSFS PSATPSQKYN SRSNRGEVVT SFGLAQGVSW SGRGGAGNIS LKVLGCPEAL TGSYKSMFQK LPDIREVLTC KIEELGSEL KEHYKIEAFT PLLAPAQEPV TLLGQIGCDS NGKLNNKSVI LEGDREHSSG AQIPVDLSEL KEYSLFPGQV V IMEGINTT ...String:
HQLLSPSSFS PSATPSQKYN SRSNRGEVVT SFGLAQGVSW SGRGGAGNIS LKVLGCPEAL TGSYKSMFQK LPDIREVLTC KIEELGSEL KEHYKIEAFT PLLAPAQEPV TLLGQIGCDS NGKLNNKSVI LEGDREHSSG AQIPVDLSEL KEYSLFPGQV V IMEGINTT GRKLVATKLY EGVPLPFYQP TEEDADFEQS MVLVACGPYT TSDSITYDPL LDLIAVINHD RPDVCILFGP FL DAKHEQV ENCLLTSPFE DIFKQCLRTI IEGTRSSGSH LVFVPSLRDV HHEPVYPQPP FSYSDLSRED KKQVQFVSEP CSL SINGVI FGLTSTDLLF HLGAEEISSS SGTSDRFSRI LKHILTQRSY YPLYPPQEDM AIDYESFYVY AQLPVTPDVL IIPS ELRYF VKDVLGCVCV NPGRLTKGQV GGTFARLYLR RPAADGAERQ SPCIAVQVVR I

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*G)-3')

MacromoleculeName: DNA (5'-D(P*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')
type: dna / ID: 8
Details: Residue 1-35 forms the DNA double-stranded hairpin foldback. The rest of the DNA forms the single-stranded DNA template for the enzyme to bind.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 4.720067 KDa
SequenceString:
(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

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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Map obtained from merging three independently processed and refined domains.
Number images used: 159847
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-8d0b:
Human CST-DNA polymerase alpha/primase preinitiation complex bound to 4xTEL-foldback template

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