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- EMDB-26867: Tetrahymena Polymerase alpha-Primase -

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Basic information

Entry
Database: EMDB / ID: EMD-26867
TitleTetrahymena Polymerase alpha-Primase
Map datafull map
Sample
  • Complex: Tetrahymena PolaPrim
    • Protein or peptide: DNA polymerase alpha subunit BDNA polymerase
    • Protein or peptide: DNA polymerase
    • Protein or peptide: Eukaryotic-type DNA primase, large subunit
    • Protein or peptide: DNA primasePrimase
  • Ligand: ZINC ION
KeywordsCST / polymerase / primase / REPLICATION
Function / homology
Function and homology information


DNA primase activity / mitotic DNA replication initiation / DNA replication, synthesis of primer / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding ...DNA primase activity / mitotic DNA replication initiation / DNA replication, synthesis of primer / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / metal ion binding / nucleus
Similarity search - Function
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 / Eukaryotic and archaeal DNA primase, large subunit / DNA polymerase alpha catalytic subunit, catalytic domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B ...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 / Eukaryotic and archaeal DNA primase, large subunit / DNA polymerase alpha catalytic subunit, catalytic domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb 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-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase alpha subunit B / DNA polymerase / Eukaryotic-type DNA primase, large subunit / DNA primase
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsHe Y / Song H / Chan H / Wang Y / Liu B / Susac L / Zhou ZH / Feigon J
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131901 United States
National Science Foundation (NSF, United States)MCB2016540 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071940 United States
CitationJournal: Nature / Year: 2022
Title: Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.
Authors: Yao He / He Song / Henry Chan / Baocheng Liu / Yaqiang Wang / Lukas Sušac / Z Hong Zhou / Juli Feigon /
Abstract: Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single- ...Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand and TPP1 (in complex with TIN2) recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The telomere DNA ends are replicated and maintained by telomerase, for the G-strand, and subsequently DNA polymerase α-primase (PolαPrim), for the C-strand. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN1 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.
History
DepositionMay 6, 2022-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26867.png

Downloads & links

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Map

FileDownload / File: emd_26867.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.013
Minimum - Maximum-0.0107943965 - 0.04020112
Average (Standard dev.)0.00015049666 (±0.0016082253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26867_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: composite map generated using two focused refined maps, sharpened

Fileemd_26867_additional_1.map
Annotationcomposite map generated using two focused refined maps, sharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half1

Fileemd_26867_half_map_1.map
Annotationhalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half2

Fileemd_26867_half_map_2.map
Annotationhalf2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetrahymena PolaPrim

EntireName: Tetrahymena PolaPrim
Components
  • Complex: Tetrahymena PolaPrim
    • Protein or peptide: DNA polymerase alpha subunit BDNA polymerase
    • Protein or peptide: DNA polymerase
    • Protein or peptide: Eukaryotic-type DNA primase, large subunit
    • Protein or peptide: DNA primasePrimase
  • Ligand: ZINC ION

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Supramolecule #1: Tetrahymena PolaPrim

SupramoleculeName: Tetrahymena PolaPrim / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Tetrahymena thermophila (eukaryote)

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Macromolecule #1: DNA polymerase alpha subunit B

MacromoleculeName: DNA polymerase alpha subunit B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 68.773758 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEEFEKILEE IDSANLSQNA VQQLKQLIIS NGILDDEQLM EKWLFQLEAI IYNSKQKFLE AQLMDFKKNV LNQDIKKPQN DNFKKIKES EVQKNIDIIK GDADLFTKAS SAKKQTSTQQ ANNAAIDCEE QLRQQLGKES MLDPKRFSYN SNLPQVNFEK N QHQNSNNM ...String:
MEEFEKILEE IDSANLSQNA VQQLKQLIIS NGILDDEQLM EKWLFQLEAI IYNSKQKFLE AQLMDFKKNV LNQDIKKPQN DNFKKIKES EVQKNIDIIK GDADLFTKAS SAKKQTSTQQ ANNAAIDCEE QLRQQLGKES MLDPKRFSYN SNLPQVNFEK N QHQNSNNM TIVDLFEQYD PNKIPNYLTN NIDKIRKHLE QRILTFKIQN YSQFIVNGEQ LINDVSSYSK TEEVKMVGRL IS TENGFLN TTNLRIELNR NQYFDLVFEN DFDFGNNVLF PNQIVMVKGI INEKEELVVS ELITDHIKEI TDEEHPKIDN LNQ DESQLI MVAAGPFSTV MSTQYTSFDN ILHVAKTKKV STLILLGPFL DIKNEILKDG SIIINSKEYT FEQLQNSLFE KAVK ELEGK TQIIIVPSTR DILSTDSIPQ MSLEIKVSEH KNSIHSYSNP AYIDIDKLRV YIANSDVGMI TLQNSLLDKK IPYMQ QSRL TFKALMNQQN LYSIYPMRNP QDSQQILETV KLPNINDFDI PFDYQLEQYP HIIISPSSLP KFATKIYNTV VINPNY VIR DGSQAGNFAI ITLFKDSDIP IHERTRVDLY CL

UniProtKB: DNA polymerase alpha subunit B

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Macromolecule #2: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 161.986984 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSDKLTRLER LNKEVKKQNK LKQHSKNNRF DDDMDIEAYE DDEQIEEDDF IDDTQEDKKY KKKYREIEDE FDQEIEEEEE LNKKKKTKN TILNYTNTTA VTNNKKKAIS KQIPDIDIEE IMKLTERKKK IEQEEAQLLQ EEQELLEQEK REEEEKKRIS Q EAKSILRE ...String:
MSDKLTRLER LNKEVKKQNK LKQHSKNNRF DDDMDIEAYE DDEQIEEDDF IDDTQEDKKY KKKYREIEDE FDQEIEEEEE LNKKKKTKN TILNYTNTTA VTNNKKKAIS KQIPDIDIEE IMKLTERKKK IEQEEAQLLQ EEQELLEQEK REEEEKKRIS Q EAKSILRE DCASSKTANS SKGKVDQNIL NAINRDFSDD SNTVDSISEF QKLKSLAQKA NLANESLKQS KVSNTEINLT NL SISQVKK INDYKNEDGS VDAYLYDYFY DAQVKPDKIY AFAKVQNKQT NAFDTCVIQI DTIIRNLFFY PSSDTVTEQQ IKN EIAELL KKEQTSRKNV EFLGAFVDKN YAFELPIPRG KSRWYQVVMS YEYEVISPDT KGQYFSYCVG STYSALETFL ITKK ITGPS WVRFQNVKDT TSCITNRKLE FRVDYTNQSN IQVLQKQLPT PPLSVVCISL KTSQQIVLSQ KKKEYKKEIF NLNMK YHEG INIDNSNKDE LNQFKSISFI THIDPTKKQD SITKKGTLPE TTKFCLNELN LLEQFLVHFN EIDPDIVVAH DLYSTV FEI ILTRIREKGI RKWNLLSKLI NIGSSDIPKY GSSTFKTKMA MKGRLLVDTL LSSQEFVNCV EYTLEALAQK LFKIEIP RI DAKAYQQKFA TYKLLNSLVD DTYQDIDYAL RIMYHLQIVP LTKQLTSICG NIWMGSLQNQ RAERNEMLLL HKFNQLNY V YPDNFKNLPE SYKKKHKNAQ IRKQYEEDED QAQGNKNPKK KENKYKGGQV FEPEKGLYNE YIVLLDFNSL YPSIIQEFN VCFTTCVRDP IPLEMQMAPF LGNKKAAIQY SKNQNTKENK MQDEDEEDNE NEQIVQTHDV LPTIEVIKGI APLPSILQYL VEQRKVVKN QIKGQKDPQV IETLDIKQKA FKLVANSMYG CLGFSSSRFY AMPLASFITA KGRHILFDSK KIVEDMGYSV I YGDTDSLM IKPGTNEFLE AVKTGLSIKI KVNSKYKKLQ LDIDGVFKNM LLLKKKKYAT LKVANWEEVK NTNAPEKLEK EI KGIDVVR RDWCQLSRDA GNKILEIILE SKSSENMLDD IKKYLIQLND DINQKNIKNS NYYITKRLTK RVDQYGEKNL PHV AVAQRS IQEKGIDPQT YVNQIISYII CKNEQSSRLV DKAYSPQEFI TQSKSLEIDL QYYKRFQLFE PIKRMLEVIE GINL QEIAS ILEVHYSVQH VSQNNELNAE NVLNLKSKRN QFLTSIPRVL VDCKKCDQTF LFLGILEENA DAASILKCKC GNDIY IQLK NKIALVVKEL IRNFEENAIQ IDNEEFEYTH QISLVGKAKQ QKMSSFTLNQ KLLSIQAMFD ITKEEQENTQ KVTIEK IKT IKKTLDDLLS KSQYNNLNLS NIFTSFGLLK

UniProtKB: DNA polymerase

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Macromolecule #3: Eukaryotic-type DNA primase, large subunit

MacromoleculeName: Eukaryotic-type DNA primase, large subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 64.386645 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MINNKQIQKI QQISKFQLKE YIEMQMQQHS LNKQIKEEER NYILLENAID NSNIQGFQTI QEIVQLAMQR MKMIKEIIQE EPRFNLIDP LSKPNYNRIR VANKFITVAI PKSDPEYELK NKQQYAKEVL SHFLCKMAYA FYAEPETWLE FARAEAFILM D KLKSGQHH ...String:
MINNKQIQKI QQISKFQLKE YIEMQMQQHS LNKQIKEEER NYILLENAID NSNIQGFQTI QEIVQLAMQR MKMIKEIIQE EPRFNLIDP LSKPNYNRIR VANKFITVAI PKSDPEYELK NKQQYAKEVL SHFLCKMAYA FYAEPETWLE FARAEAFILM D KLKSGQHH ANFFSDENLK IKTISDELFK QAFPKIEATF SSIKIKKNDS EVEQINIKKD NFKMFKFIDH PSMLSNNDVV LH KGYIIIY KESTSKIVQN IFIERLLDEM RLLQLKFQNN GSKLDDDRLS FLKDLHKAEI FNDSTQFNST QIHHYELDRL AKR DMPACM TYLMYGLNQK LHLKHFGRLQ LGLFLKGAGL SLNEALTFWQ KKFSKTSADD FKKKYDYNIR HNYGKLGKQL DYTP MSCQK IIGFQPLKDE FHGCPYKTME SQQLKDFLKL SYNITDEQFV QVNIFKNQKQ YQLACKEVFK VLNDTRDKTK EQFYP YFDK VGNHPNAYFE QSLRMHEPQR FQKQDEEKKQ NKQNRNQNFS ANKQSTNKNN QMDLEF

UniProtKB: Eukaryotic-type DNA primase, large subunit

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Macromolecule #4: DNA primase

MacromoleculeName: DNA primase / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 47.172855 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEIETVEVAP QQEEEFKLDY DLLEEYYRSY FPVSQMVQWL SYPQDGDDTY FTRREFSFTL QNEVYLRYQM YNSEREFKNA LLQKVPEKI DIGAVYDRPG KKGDDIKAKE KEFVIDIDMT DYDHIRTCCS KAKICEKCWK FMRVACDLIS KSLDEDFGFQ H VLWVYSGR ...String:
MEIETVEVAP QQEEEFKLDY DLLEEYYRSY FPVSQMVQWL SYPQDGDDTY FTRREFSFTL QNEVYLRYQM YNSEREFKNA LLQKVPEKI DIGAVYDRPG KKGDDIKAKE KEFVIDIDMT DYDHIRTCCS KAKICEKCWK FMRVACDLIS KSLDEDFGFQ H VLWVYSGR RGIHAWVCDK EIRKANDYTR ASIIDYLNIL VDNSIGSSYV KPSLLKMEKS HLIERNAMKQ LNQKNDDLKA EK ESEKVFV EIVLREQNLF MKKPEIILEF LAKRSENLSK EVEKEWKTLK TSEQRYEALK ELVSSEDKKK THYLLEELRI WLL YPRLDV NVSKSTNHLL KSPFCIHPKT GNVCVPFTTE EISTFDPFSV PNISTLTTEE GSSKMKNSLK IFNKFLENLK KDV

UniProtKB: DNA primase

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 264498
FSC plot (resolution estimation)

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