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- EMDB-15306: Pol12, PolA1 and Pri2 N-terminal domain local refinement. Pol alp... -

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Basic information

Entry
Database: EMDB / ID: EMD-15306
TitlePol12, PolA1 and Pri2 N-terminal domain local refinement. Pol alpha - replisome complex.
Map dataPol12/PolA1/Pri2 N-term domain local refinement - locally filtered
Sample
  • Complex: Replisome - pol alpha complex
KeywordsReplication / helicase / polymerase / pol alpha / priming
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.04 Å
AuthorsJones ML / Yeeles JTP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_1201/12 United Kingdom
CitationJournal: Mol Cell / Year: 2023
Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication.
Authors: Morgan L Jones / Valentina Aria / Yasemin Baris / Joseph T P Yeeles /
Abstract: During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand ...During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
History
DepositionJul 4, 2022-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15306.png

Downloads & links

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Map

FileDownload / File: emd_15306.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPol12/PolA1/Pri2 N-term domain local refinement - locally filtered
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.27 Å/pix.
x 220 pix.
= 498.806 Å		2.27 Å/pix.
x 220 pix.
= 498.806 Å		2.27 Å/pix.
x 220 pix.
= 498.806 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2673 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.77
Minimum - Maximum-23.019556000000001 - 48.885795999999999
Average (Standard dev.)0.0031307724 (±0.14105642)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 498.80597 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Pol12/PolA1/Pri2 N-term domain local refinement - unsharpened

Fileemd_15306_additional_1.map
AnnotationPol12/PolA1/Pri2 N-term domain local refinement - unsharpened
Projections & Slices
AxesZYX

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Half map: half map A

Fileemd_15306_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_15306_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Replisome - pol alpha complex

EntireName: Replisome - pol alpha complex
Components
  • Complex: Replisome - pol alpha complex

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Supramolecule #1: Replisome - pol alpha complex

SupramoleculeName: Replisome - pol alpha complex / type: complex / ID: 1 / Parent: 0
Details: S. cerevisiae pol alpha bound to the core replisome engaged with a fork DNA substrate containing a 60 nucleotide lagging strand.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.184 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 100178
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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