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- EMDB-26866: Tetrahymena CST with Polymerase alpha-Primase -

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Basic information

Entry
Database: EMDB / ID: EMD-26866
TitleTetrahymena CST with Polymerase alpha-Primase
Map datafull map
Sample
  • Complex: Tetrahymena CST-PolaPrim
    • Complex: Tetrahymena CST
      • Protein or peptide: Telomerase-associated protein of 75 kDa
      • Protein or peptide: Telomerase-associated protein of 45 kDa
      • Protein or peptide: Telomerase-associated protein of 19 kDa
      • Protein or peptide: Telomerase associated protein p50
      • DNA: Telomere DNA
    • Complex: Tetrahymena DNA polymerase alpha-primase
      • Protein or peptide: DNA polymerase
  • Ligand: ZINC ION
Keywordstelomerase / RNP / CST / polymerase / REPLICATION
Function / homology
Function and homology information


mitotic DNA replication initiation / telomerase holoenzyme complex / telomere maintenance via telomerase / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / metal ion binding / nucleus
Similarity search - Function
DNA polymerase alpha catalytic subunit, catalytic domain / 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 polymerase alpha catalytic subunit, catalytic domain / 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
Telomerase-associated protein of 75 kDa / Telomerase-associated protein of 19 kDa / Telomerase-associated protein of 50 kDa / DNA polymerase / Telomerase-associated protein of 45 kDa
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 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_26866.png

Downloads & links

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Map

FileDownload / File: emd_26866.map.gz / Format: CCP4 / Size: 244.1 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-0.025084842 - 0.06263634
Average (Standard dev.)0.000097714226 (±0.001549412)
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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Mask #1

Fileemd_26866_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half1

Fileemd_26866_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_26866_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 CST-PolaPrim

EntireName: Tetrahymena CST-PolaPrim
Components
  • Complex: Tetrahymena CST-PolaPrim
    • Complex: Tetrahymena CST
      • Protein or peptide: Telomerase-associated protein of 75 kDa
      • Protein or peptide: Telomerase-associated protein of 45 kDa
      • Protein or peptide: Telomerase-associated protein of 19 kDa
      • Protein or peptide: Telomerase associated protein p50
      • DNA: Telomere DNA
    • Complex: Tetrahymena DNA polymerase alpha-primase
      • Protein or peptide: DNA polymerase
  • Ligand: ZINC ION

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Supramolecule #1: Tetrahymena CST-PolaPrim

SupramoleculeName: Tetrahymena CST-PolaPrim / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6

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Supramolecule #2: Tetrahymena CST

SupramoleculeName: Tetrahymena CST / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4, #6
Source (natural)Organism: Tetrahymena thermophila (eukaryote)

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Supramolecule #3: Tetrahymena DNA polymerase alpha-primase

SupramoleculeName: Tetrahymena DNA polymerase alpha-primase / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Tetrahymena thermophila (eukaryote)

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Macromolecule #1: Telomerase-associated protein of 75 kDa

MacromoleculeName: Telomerase-associated protein of 75 kDa / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 73.899602 KDa
SequenceString: MEIEEDLNLK ILEDVKKLYL QSFDYIKNGI SSSLPSDKKF LADDDIDLSR ITFLYKFISV NPTLLLINEK TQAKRRIFQG EYLYGKKKI QFNIIAKNLE IERELIQFFK KPYQCYIMHN VQVFQMLNKN KNNNVVEFMD SEDLQSSVDC QLYYLIDESS H VLEDDSMD ...String:
MEIEEDLNLK ILEDVKKLYL QSFDYIKNGI SSSLPSDKKF LADDDIDLSR ITFLYKFISV NPTLLLINEK TQAKRRIFQG EYLYGKKKI QFNIIAKNLE IERELIQFFK KPYQCYIMHN VQVFQMLNKN KNNNVVEFMD SEDLQSSVDC QLYYLIDESS H VLEDDSMD FISTLTRLSD SFNSNEFVFE TNYSIQISQM PKPLNTTHFK LLQPKVVNSF EGVILQVQEG KNILQIEELI DQ VYLNSRR DRFYILKVAN GKNYMDFIEV YLVYDNEDQE AKQQLQFYLK PFQRILIFQS LKHFTKNLKL FMISFFYSSG VQP NNSNVK NFLVSHKGVE FFSRFDIQKN ELLCKDLIKS YNKLPLSNIS KLLEDEGVMI RSNMKFQVRV KKVKYFKIRL NCLN CKQEW TVGLKNCINC KGQQSYISYN IQVLVQDQHF LEQQAYIYLY DDLAAQFFNI TESEKKELHL HLTKNETFIQ LYYSF NKDY PLSIIKFKDK IFNKDITNCI VAYPFADIDN KIFNSQQQII QDENLRIESE KFIQNFTEDN NLQESKLYYE KFKSKN KQQ IFVNGTYIST NYSQGQKICL KPIPCLKVMY VFPQEDIKLS ALKIIEEINQ LKIQIDQLN

UniProtKB: Telomerase-associated protein of 75 kDa

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Macromolecule #2: Telomerase-associated protein of 45 kDa

MacromoleculeName: Telomerase-associated protein of 45 kDa / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 43.682938 KDa
SequenceString: MEDNFELVFL KELPSLPDFS KVCFTGLILS FSNFPSSEQN QQKDVPHKIA IIQDSTGEAE LFLDMYKFCQ EEISVFKAIT GIGVLKKKN IGAGQVCKII VERFRIIHSA DEEMLQYLLI QKYKLSKTLN EQQQIKQKEQ QINQQKIDKV VQDKESKEHL L WKQQQIPQ ...String:
MEDNFELVFL KELPSLPDFS KVCFTGLILS FSNFPSSEQN QQKDVPHKIA IIQDSTGEAE LFLDMYKFCQ EEISVFKAIT GIGVLKKKN IGAGQVCKII VERFRIIHSA DEEMLQYLLI QKYKLSKTLN EQQQIKQKEQ QINQQKIDKV VQDKESKEHL L WKQQQIPQ IKSNQENINT LKYKELIAGE LMRITHKLLI QKLQQQQPAN NNKQINEMDV ESNELAEKKE VIIKIQEIAK DQ QLYDTLS IQYQVDQKEQ YYAKIAQSLE DFVSISALKM VSYIYPNISY QVSIGFFQNI LDIATKTVKD RGALGCNYKY LKD KLTKAL NLQQISYPLI SESYISYLVH LFQDFNIIEI ENEHKFYYKQ AFQYDDS

UniProtKB: Telomerase-associated protein of 45 kDa

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Macromolecule #3: Telomerase-associated protein of 19 kDa

MacromoleculeName: Telomerase-associated protein of 19 kDa / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 19.498049 KDa
SequenceString:
MQQPKRNFDL YKLITDKQID FQVADLIQDE QSSFVSVRIY GQFKCFVPKS TIQEQLDKIK NLSSKELAKN KIFKFLSEYN KNNQKQDEL SHDYYGYFKV QQHQFILNLE NAQREASLAV DDFYFINGRI YKTNHDILIL QAHHVYQMQK PTLQLLQAAS E INQN

UniProtKB: Telomerase-associated protein of 19 kDa

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Macromolecule #4: Telomerase associated protein p50

MacromoleculeName: Telomerase associated protein p50 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 50.049688 KDa
SequenceString: MKLLLQNQNI FQKLKNTLNG CIKKFYDTYQ DLEQMQKFEM IVEDKLLFRY SCSQSEMFSA QIQAHYLEKR VLQLTDGNVK YIVNFRDKG VLDKANFFDT PNNSLVIIRQ WSYEIYYTKN TFQINLVIDE MRCIDIITTI FYCKLELDFT QGIKGISKSS S FSNQIYEY ...String:
MKLLLQNQNI FQKLKNTLNG CIKKFYDTYQ DLEQMQKFEM IVEDKLLFRY SCSQSEMFSA QIQAHYLEKR VLQLTDGNVK YIVNFRDKG VLDKANFFDT PNNSLVIIRQ WSYEIYYTKN TFQINLVIDE MRCIDIITTI FYCKLELDFT QGIKGISKSS S FSNQIYEY SAQYYKAIQL LKKLLINDSY ISELYNSTKS KQQPRLFIFQ SFKPKMNLAE QNLSRQFEQC QQDDFGDGCL LQ IVNYTHQ SLKQIENKNN SNQIVNGQNE ISKKKRVLKS NEDLYKISLQ KQLKIFQEEE IELHSQSTIR NQTNQQLETF ESD TSKRNS EKILHSINEL NTSKQKVNQM NSSQHQIQKL ENNNLNKNIL NQINENDIKN ELEERQQQHL TQSFNSKAQL KKII TLKKN QDILLFKPQE QEGSKKY

UniProtKB: Telomerase-associated protein of 50 kDa

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Macromolecule #5: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 5 / 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 #6: Telomere DNA

MacromoleculeName: Telomere DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 19.207121 KDa
SequenceString: (DG)(DT)(DT)(DG)(DG)(DG)(DG)(DT)(DT)(DG) (DG)(DG)(DG)(DT)(DT)(DG)(DG)(DG)(DG)(DT) (DT)(DG)(DG)(DG)(DG)(DT)(DT)(DG)(DG) (DG)(DG)(DT)(DT)(DG)(DG)(DG)(DG)(DT)(DT) (DG) (DG)(DG)(DG)(DT)(DT)(DG) ...String:
(DG)(DT)(DT)(DG)(DG)(DG)(DG)(DT)(DT)(DG) (DG)(DG)(DG)(DT)(DT)(DG)(DG)(DG)(DG)(DT) (DT)(DG)(DG)(DG)(DG)(DT)(DT)(DG)(DG) (DG)(DG)(DT)(DT)(DG)(DG)(DG)(DG)(DT)(DT) (DG) (DG)(DG)(DG)(DT)(DT)(DG)(DG)(DG) (DG)(DT)(DT)(DG)(DG)(DG)(DG)(DT)(DT)(DG) (DG)(DG)

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / 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: 8
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: EMDB MAP
EMDB ID:

23437

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 142912
FSC plot (resolution estimation)

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