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- EMDB-15341: Human replisome bound by pol alpha, engaged on fork DNA containin... -

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Basic information

Entry
Database: EMDB / ID: EMD-15341
TitleHuman replisome bound by pol alpha, engaged on fork DNA containing a 60 nt lagging strand.
Map dataHuman replisome bound by pol alpha, engaged on fork DNA containing a 60 nt lagging strand.
Sample
  • Complex: Replisome - pol alpha complex
KeywordsReplication / helicase / polymerase / pol alpha / priming
Function / homology
Function and homology information


cellular response to bleomycin / DNA primase AEP / DNA secondary structure binding / ribonucleotide binding / detection of abiotic stimulus / regulation of nuclear cell cycle DNA replication / replication fork arrest / Switching of origins to a post-replicative state / cell cycle phase transition / Unwinding of DNA ...cellular response to bleomycin / DNA primase AEP / DNA secondary structure binding / ribonucleotide binding / detection of abiotic stimulus / regulation of nuclear cell cycle DNA replication / replication fork arrest / Switching of origins to a post-replicative state / cell cycle phase transition / Unwinding of DNA / DNA replication initiation / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / activation of protein kinase activity / Telomere C-strand synthesis initiation / DNA/RNA hybrid binding / cellular response to cisplatin / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / replication fork protection complex / nuclear origin of replication recognition complex / cellular response to hydroxyurea / regulation of type I interferon production / alpha DNA polymerase:primase complex / anaphase-promoting complex binding / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / mitotic DNA replication / Polymerase switching / CMG complex / DNA replication checkpoint signaling / regulation of phosphorylation / Processive synthesis on the lagging strand / DNA replication preinitiation complex / MCM complex / Removal of the Flap Intermediate / lagging strand elongation / mitotic DNA replication checkpoint signaling / double-strand break repair via break-induced replication / Polymerase switching on the C-strand of the telomere / mitotic DNA replication initiation / DNA replication, synthesis of primer / mitotic intra-S DNA damage checkpoint signaling / regulation of DNA-templated DNA replication initiation / inner cell mass cell proliferation / DNA strand elongation involved in DNA replication / Apoptotic cleavage of cellular proteins / branching morphogenesis of an epithelial tube / DNA synthesis involved in DNA repair / positive regulation of double-strand break repair / G1/S-Specific Transcription / leading strand elongation / mitotic G2 DNA damage checkpoint signaling / nuclear replication fork / replication fork processing / DNA replication origin binding / cochlea development / DNA replication initiation / Activation of the pre-replicative complex / Activation of ATR in response to replication stress / response to UV / positive regulation of double-strand break repair via homologous recombination / cellular response to interleukin-4 / DNA helicase activity / lung development / cellular response to epidermal growth factor stimulus / DNA damage checkpoint signaling / morphogenesis of an epithelium / Defective pyroptosis / Assembly of the pre-replicative complex / enzyme activator activity / helicase activity / circadian rhythm / regulation of circadian rhythm / peptidyl-serine phosphorylation / double-strand break repair via nonhomologous end joining / DNA-templated DNA replication / multicellular organism growth / cellular response to xenobiotic stimulus / nuclear matrix / Orc1 removal from chromatin / protein import into nucleus / DNA-directed RNA polymerase activity / cellular senescence / nuclear envelope / mitotic cell cycle / nucleosome assembly / single-stranded DNA binding / site of double-strand break / 4 iron, 4 sulfur cluster binding / Processing of DNA double-strand break ends / histone binding / DNA helicase / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / cell population proliferation / Ub-specific processing proteases / cilium / ciliary basal body
Similarity search - Function
: / WDHD1 HMG box / WDHD1 first WD40 domain / Claspin / Timeless, C-terminal / Timeless PAB domain / DNA polymerase alpha, subunit B, N-terminal domain superfamily / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 ...: / WDHD1 HMG box / WDHD1 first WD40 domain / Claspin / Timeless, C-terminal / Timeless PAB domain / DNA polymerase alpha, subunit B, N-terminal domain superfamily / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / : / DNA polymerase alpha subunit B, OB domain / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / Timeless, N-terminal / Timeless / Timeless protein / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain / DNA polymerase alpha, subunit B / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit C-terminal domain / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA replication licensing factor MCM2-like, winged-helix domain / DNA polymerase alpha catalytic subunit, catalytic domain / : / PSF2 N-terminal domain / : / : / PSF3 N-terminal domain / PSF1 C-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / GINS complex, subunit Psf3 superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / GINS complex, subunit Psf1 / GINS, helical bundle-like domain superfamily / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / : / MCM5, C-terminal domain / DNA replication licensing factor MCM7, winged helix / DNA replication licensing factor Mcm5 / MCM4, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / : / MCM3-like, winged helix domain / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / DNA polymerase family B, thumb domain / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain / High mobility group box domain superfamily / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B signature. / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Cell division control protein 45 homolog / WD repeat and HMG-box DNA-binding protein 1 / DNA polymerase alpha catalytic subunit / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA primase small subunit / DNA primase large subunit / DNA replication licensing factor MCM2 ...Cell division control protein 45 homolog / WD repeat and HMG-box DNA-binding protein 1 / DNA polymerase alpha catalytic subunit / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA primase small subunit / DNA primase large subunit / DNA replication licensing factor MCM2 / DNA polymerase alpha subunit B / DNA replication licensing factor MCM6 / DNA replication complex GINS protein PSF1 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF3 / TIMELESS-interacting protein / Claspin / Protein timeless homolog / DNA replication complex GINS protein PSF2
Similarity search - Component
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_15341.png

Downloads & links

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Map

FileDownload / File: emd_15341.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman replisome bound by pol alpha, engaged on fork DNA containing a 60 nt lagging strand.
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: 0.137
Minimum - Maximum-2.4792874 - 4.0916557
Average (Standard dev.)0.006940133 (±0.07243807)
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 A

Fileemd_15341_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_15341_half_map_2.map
AnnotationHalf 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: 3.4 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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