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- EMDB-17807: Cryo-EM structure of human DNA polymerase alpha-primase in pre-in... -

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Basic information

Entry
Database: EMDB / ID: EMD-17807
TitleCryo-EM structure of human DNA polymerase alpha-primase in pre-initiation stage 2
Map data
Sample
  • Complex: DNA polymerase alpha-primase
KeywordsDNA polymerase / complex / DNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.11 Å
AuthorsYin Z / Pellegrini L
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust221892/Z/20/Z United Kingdom
CitationJournal: FEBS J / Year: 2024
Title: CryoEM insights into RNA primer synthesis by the human primosome.
Authors: Zhan Yin / Mairi L Kilkenny / De-Sheng Ker / Luca Pellegrini /
Abstract: Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis ...Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo-electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation.
History
DepositionJul 7, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17807.png

Downloads & links

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Map

FileDownload / File: emd_17807.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 380 pix.
= 405.08 Å		1.07 Å/pix.
x 380 pix.
= 405.08 Å		1.07 Å/pix.
x 380 pix.
= 405.08 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.066 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.278
Minimum - Maximum-0.44074935 - 1.2455238
Average (Standard dev.)-0.00081148226 (±0.031471483)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 405.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17807_msk_1.map
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Half map: #1

Fileemd_17807_half_map_1.map
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Half map: #2

Fileemd_17807_half_map_2.map
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Sample components

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Entire : DNA polymerase alpha-primase

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

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Supramolecule #1: DNA polymerase alpha-primase

SupramoleculeName: DNA polymerase alpha-primase / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 287.5 KDa

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: ab-initio models are used
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.11 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 46239
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Details: ab-initio models are used
Final angle assignmentType: MAXIMUM LIKELIHOOD / Details: Non-uniform refinement
FSC plot (resolution estimation)

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