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- PDB-7uy6: Tetrahymena telomerase at 2.9 Angstrom resolution -

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Basic information

Entry
Database: PDB / ID: 7uy6
TitleTetrahymena telomerase at 2.9 Angstrom resolution
Components
  • (Telomerase holoenzyme ...) x 3
  • Telomerase La-related protein p65
  • Telomerase RNATelomerase RNA component
  • Telomerase associated protein p50
  • Telomerase reverse transcriptase
  • Telomere DNA
KeywordsREPLICATION / telomerase / RNP / reverse transcriptase
Function / homology
Function and homology information


telomerase catalytic core complex assembly / telomerase RNA stabilization / telomerase RNA reverse transcriptase activity / DNA replication factor A complex / single-stranded telomeric DNA binding / telomerase RNA binding / telomerase holoenzyme complex / telomeric DNA binding / telomere maintenance via telomerase / RNA-directed DNA polymerase ...telomerase catalytic core complex assembly / telomerase RNA stabilization / telomerase RNA reverse transcriptase activity / DNA replication factor A complex / single-stranded telomeric DNA binding / telomerase RNA binding / telomerase holoenzyme complex / telomeric DNA binding / telomere maintenance via telomerase / RNA-directed DNA polymerase / DNA recombination / DNA replication / chromosome, telomeric region / DNA repair / DNA binding / zinc ion binding / metal ion binding
Similarity search - Function
Telomerase reverse transcriptase TEN domain / Replication factor A protein 3 / Replication factor A protein 3 / xRRM domain profile. / La protein, xRRM domain / La domain containing protein / La domain / Domain in the RNA-binding Lupus La protein; unknown function / La-type HTH domain / La-type HTH domain profile. ...Telomerase reverse transcriptase TEN domain / Replication factor A protein 3 / Replication factor A protein 3 / xRRM domain profile. / La protein, xRRM domain / La domain containing protein / La domain / Domain in the RNA-binding Lupus La protein; unknown function / La-type HTH domain / La-type HTH domain profile. / Telomerase ribonucleoprotein complex - RNA binding domain / : / Telomerase reverse transcriptase / Telomerase ribonucleoprotein complex - RNA-binding domain / Telomerase ribonucleoprotein complex - RNA binding domain / Replication factor A, C-terminal / Replication factor-A C terminal domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Winged helix DNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Replication protein A 32 kDa subunit / Replication protein A 14 kDa subunit / Telomeric repeat-binding subunit 1 / Telomerase-associated protein of 50 kDa ...: / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Replication protein A 32 kDa subunit / Replication protein A 14 kDa subunit / Telomeric repeat-binding subunit 1 / Telomerase-associated protein of 50 kDa / Telomerase reverse transcriptase / La-related protein 7 homolog
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHe, Y. / Song, H. / Chan, H. / Wang, Y. / Liu, B. / Susac, L. / Zhou, Z.H. / Feigon, J.
Funding support United States, 3items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 7, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
H: Telomerase La-related protein p65
A: Telomerase reverse transcriptase
D: Telomerase holoenzyme Teb1 subunit
E: Telomerase holoenzyme Teb2 subunit
F: Telomerase holoenzyme Teb3 subunit
G: Telomerase associated protein p50
B: Telomerase RNA
C: Telomere DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)444,8049
Polymers444,7398
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules HAG

#1: Protein Telomerase La-related protein p65 / Telomerase-associated protein of 65 kDa / p65


Mass: 64207.363 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / References: UniProt: W7X6T2
#2: Protein Telomerase reverse transcriptase / / Telomerase catalytic subunit / Telomerase subunit P133


Mass: 133486.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / References: UniProt: O77448, RNA-directed DNA polymerase
#6: Protein Telomerase associated protein p50 / p50


Mass: 50049.688 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / References: UniProt: D2CVN8

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Telomerase holoenzyme ... , 3 types, 3 molecules DEF

#3: Protein Telomerase holoenzyme Teb1 subunit /


Mass: 82040.289 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / References: UniProt: D2CVN6
#4: Protein Telomerase holoenzyme Teb2 subunit /


Mass: 30993.035 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / References: UniProt: A0A0U8TRG9
#5: Protein Telomerase holoenzyme Teb3 subunit /


Mass: 14007.626 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / References: UniProt: A0A0U8UFF4

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RNA chain / DNA chain / Non-polymers , 3 types, 3 molecules BC

#7: RNA chain Telomerase RNA / Telomerase RNA component


Mass: 50746.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / References: GenBank: 15148878
#8: DNA chain Telomere DNA


Mass: 19207.121 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tetrahymena thermophila (eukaryote)
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Tetrahymena telomerase holoenzyme / Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 539078 / Symmetry type: POINT
RefinementHighest resolution: 2.9 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00219445
ELECTRON MICROSCOPYf_angle_d0.42527002
ELECTRON MICROSCOPYf_dihedral_angle_d14.7663989
ELECTRON MICROSCOPYf_chiral_restr0.0353113
ELECTRON MICROSCOPYf_plane_restr0.0032835

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