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- EMDB-26869: Tetrahymena Polymerase alpha-Primase subunits PRIM2-PRIM1 -

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Basic information

Entry
Database: EMDB / ID: EMD-26869
TitleTetrahymena Polymerase alpha-Primase subunits PRIM2-PRIM1
Map dataTetrahymena Polymerase alpha-Primase subunits PRIM2-PRIM1
Sample
  • Complex: Tetrahymena polymerase alpha-primase
Keywordspolymerase / primase / DNA / REPLICATION
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 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-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26869.png

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Map

FileDownload / File: emd_26869.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTetrahymena Polymerase alpha-Primase subunits PRIM2-PRIM1
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 8.0
Minimum - Maximum-25.326505999999998 - 35.695120000000003
Average (Standard dev.)-0.000000000004328 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
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_26869_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Tetrahymena Polymerase alpha-Primase subunits PRIM2-PRIM1

Fileemd_26869_half_map_1.map
AnnotationTetrahymena Polymerase alpha-Primase subunits PRIM2-PRIM1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Tetrahymena Polymerase alpha-Primase subunits PRIM2-PRIM1

Fileemd_26869_half_map_2.map
AnnotationTetrahymena Polymerase alpha-Primase subunits PRIM2-PRIM1
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 polymerase alpha-primase

EntireName: Tetrahymena polymerase alpha-primase
Components
  • Complex: Tetrahymena polymerase alpha-primase

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Supramolecule #1: Tetrahymena polymerase alpha-primase

SupramoleculeName: Tetrahymena polymerase alpha-primase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Tetrahymena thermophila (eukaryote)

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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: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 264498
FSC plot (resolution estimation)

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