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- EMDB-15918: Human replisome + pol alpha. Not engaged on DNA. -

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Basic information

Entry
Database: EMDB / ID: EMD-15918
TitleHuman replisome + pol alpha. Not engaged on DNA.
Map dataReplisome prepared with fork DNA substrate containing 15 nucleotide lagging strand pol alpha, complex off DNA. Unsharpened map
Sample
  • Complex: Replisome - pol alpha complex
KeywordsReplication / helicase / polymerase / pol alpha / priming
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 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
DepositionOct 4, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15918.png

Downloads & links

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Map

FileDownload / File: emd_15918.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReplisome prepared with fork DNA substrate containing 15 nucleotide lagging strand pol alpha, complex off DNA. Unsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 420 pix.
= 449.4 Å		1.07 Å/pix.
x 420 pix.
= 449.4 Å		1.07 Å/pix.
x 420 pix.
= 449.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.552
Minimum - Maximum-1.2445647 - 3.13596
Average (Standard dev.)0.004675608 (±0.11770627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 449.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Replisome prepared with fork DNA substrate containing 15...

Fileemd_15918_additional_1.map
AnnotationReplisome prepared with fork DNA substrate containing 15 nucleotide lagging strand pol alpha, complex off DNA. Local filtered, binned.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Replisome prepared with fork DNA substrate containing 15...

Fileemd_15918_additional_2.map
AnnotationReplisome prepared with fork DNA substrate containing 15 nucleotide lagging strand pol alpha, complex off DNA. Sharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Replisome prepared with fork DNA substrate containing 15...

Fileemd_15918_half_map_1.map
AnnotationReplisome prepared with fork DNA substrate containing 15 nucleotide lagging strand pol alpha, complex off DNA. Half map A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Replisome prepared with fork DNA substrate containing 15...

Fileemd_15918_half_map_2.map
AnnotationReplisome prepared with fork DNA substrate containing 15 nucleotide lagging strand pol alpha, complex off DNA. Half map B.
Projections & Slices
AxesZYX

Projections

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: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 28202
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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