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- EMDB-17198: Cryo-EM density map of the C. elegans complex CMG helicase/TIM-1/... -

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Entry
Database: EMDB / ID: EMD-17198
TitleCryo-EM density map of the C. elegans complex CMG helicase/TIM-1/TIPN-1/DNSN-1 dimer on fork DNA (prior to enrichment of particles containing TIM-1/TIPN-1)
Map dataCryo-EM density map of the C. elegans complex CMG helicase/TIM-1/TIPN-1/DNSN-1 dimer on fork DNA (prior to enrichment of particles containing TIM-1/TIPN-1)
Sample
  • Complex: In vitro reconstituted complex of Caenorhabditis elegans CMG helicase bound by TIM-1/TIPN-1 and a homodimer of DNSN-1, on a synthetic forked DNA substrate
KeywordsReplisome / DONSON / DNA replication / Initiation / REPLICATION
Biological speciesCaenorhabditis elegans (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsJenkyn-Bedford M / Yeeles JTP
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) United Kingdom
Cancer Research UK United Kingdom
CitationJournal: Science / Year: 2023
Title: DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in .
Authors: Yisui Xia / Remi Sonneville / Michael Jenkyn-Bedford / Liqin Ji / Constance Alabert / Ye Hong / Joseph T P Yeeles / Karim P M Labib /
Abstract: Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that ...Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.
History
DepositionApr 25, 2023-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17198.png

Downloads & links

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Map

FileDownload / File: emd_17198.map.gz / Format: CCP4 / Size: 120.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM density map of the C. elegans complex CMG helicase/TIM-1/TIPN-1/DNSN-1 dimer on fork DNA (prior to enrichment of particles containing TIM-1/TIPN-1)
Voxel sizeX=Y=Z: 1.295 Å
Density
Contour LevelBy AUTHOR: 0.0272
Minimum - Maximum-0.06506705 - 0.16113526
Average (Standard dev.)-0.00010829858 (±0.0033751857)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions316316316
Spacing316316316
CellA=B=C: 409.22 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map 1. Cryo-EM density map of the C....

Fileemd_17198_half_map_1.map
AnnotationHalf-map 1. Cryo-EM density map of the C. elegans complex CMG helicase/TIM-1/TIPN-1/DNSN-1 dimer on fork DNA (prior to enrichment of particles containing TIM-1/TIPN-1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2. Cryo-EM density map of the C....

Fileemd_17198_half_map_2.map
AnnotationHalf-map 2. Cryo-EM density map of the C. elegans complex CMG helicase/TIM-1/TIPN-1/DNSN-1 dimer on fork DNA (prior to enrichment of particles containing TIM-1/TIPN-1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : In vitro reconstituted complex of Caenorhabditis elegans CMG heli...

EntireName: In vitro reconstituted complex of Caenorhabditis elegans CMG helicase bound by TIM-1/TIPN-1 and a homodimer of DNSN-1, on a synthetic forked DNA substrate
Components
  • Complex: In vitro reconstituted complex of Caenorhabditis elegans CMG helicase bound by TIM-1/TIPN-1 and a homodimer of DNSN-1, on a synthetic forked DNA substrate

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Supramolecule #1: In vitro reconstituted complex of Caenorhabditis elegans CMG heli...

SupramoleculeName: In vitro reconstituted complex of Caenorhabditis elegans CMG helicase bound by TIM-1/TIPN-1 and a homodimer of DNSN-1, on a synthetic forked DNA substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#16
Details: All Caenorhabditis elegans proteins recombinantly expressed in either Saccharomyces cerevisiae (CMG helicase, TIM-1/TIPN-1) or Escherichia coli Rosetta (DE3) Competent cells (DNSN-1)
Source (natural)Organism: Caenorhabditis elegans (invertebrata)

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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
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec. / Details: PELCO easiGlow. 15 mA
VitrificationCryogen name: ETHANE / Details: Manual plunger.

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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: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 10825 / Average exposure time: 1.7 sec. / Average electron dose: 40.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4540000
Startup modelType of model: EMDB MAP
EMDB ID:

10619

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 170000

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