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- EMDB-15342: AND-1, CDC45, GINS local refinement. Human replisome bound by pol... -

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Basic information

Entry
Database: EMDB / ID: EMD-15342
TitleAND-1, CDC45, GINS local refinement. Human replisome bound by pol alpha - engaged on a 60 nt lagging strand DNA fork substrate.
Map dataAND-1, CDC45, GINS, local refinement, locally filtered. Human replisome bound by pol alpha 60 nt lagging strand fork DNA substrate.
Sample
  • Complex: Replisome - pol alpha complex
KeywordsReplication / helicase / polymerase / pol alpha / priming
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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 5, 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_15342.png

Downloads & links

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Map

FileDownload / File: emd_15342.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAND-1, CDC45, GINS, local refinement, locally filtered. Human replisome bound by pol alpha 60 nt lagging strand fork DNA substrate.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.24 Å/pix.
x 320 pix.
= 395.616 Å		1.24 Å/pix.
x 320 pix.
= 395.616 Å		1.24 Å/pix.
x 320 pix.
= 395.616 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2363 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.02
Minimum - Maximum-0.73523223 - 2.7785788
Average (Standard dev.)0.008859233 (±0.13616139)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 395.616 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_15342_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_15342_half_map_2.map
AnnotationHalf map A
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: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 174696
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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