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- EMDB-13457: cryoSPARC refinement of core human replisome displaying lagging s... -

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Basic information

Entry
Database: EMDB / ID: EMD-13457
TitlecryoSPARC refinement of core human replisome displaying lagging strand density
Map data
Sample
  • Complex: Core human replisome
Function / homology
Function and homology information


cellular response to bleomycin / DNA secondary structure binding / Switching of origins to a post-replicative state / detection of abiotic stimulus / replication fork arrest / regulation of nuclear cell cycle DNA replication / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / cell cycle phase transition ...cellular response to bleomycin / DNA secondary structure binding / Switching of origins to a post-replicative state / detection of abiotic stimulus / replication fork arrest / regulation of nuclear cell cycle DNA replication / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / cell cycle phase transition / cellular response to cisplatin / GINS complex / DNA strand elongation involved in mitotic DNA replication / mitotic DNA replication preinitiation complex assembly / nuclear origin of replication recognition complex / cellular response to hydroxyurea / alpha DNA polymerase:primase complex / mitotic DNA replication / anaphase-promoting complex binding / CMG complex / DNA replication checkpoint signaling / single-stranded 3'-5' DNA helicase activity / entrainment of circadian clock / single-stranded DNA 3'-5' DNA exonuclease activity / regulation of phosphorylation / MCM complex / DNA replication preinitiation complex / replication fork protection complex / mitotic DNA replication checkpoint signaling / mitotic DNA replication initiation / double-strand break repair via break-induced replication / mitotic intra-S DNA damage checkpoint signaling / positive regulation of double-strand break repair / regulation of DNA-templated DNA replication initiation / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / single-stranded DNA helicase activity / inner cell mass cell proliferation / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / DNA strand elongation involved in DNA replication / branching morphogenesis of an epithelial tube / DNA synthesis involved in DNA repair / cochlea development / G1/S-Specific Transcription / activation of protein kinase activity / leading strand elongation / replication fork processing / Apoptotic cleavage of cellular proteins / DNA unwinding involved in DNA replication / nuclear replication fork / mitotic G2 DNA damage checkpoint signaling / 3'-5' DNA helicase activity / DNA replication origin binding / positive regulation of double-strand break repair via homologous recombination / Activation of the pre-replicative complex / PCNA-Dependent Long Patch Base Excision Repair / DNA replication initiation / embryonic organ development / cellular response to interleukin-4 / error-prone translesion synthesis / Activation of ATR in response to replication stress / response to UV / Gap-filling DNA repair synthesis and ligation in GG-NER / base-excision repair, gap-filling / ciliary basal body / DNA damage checkpoint signaling / Assembly of the pre-replicative complex / morphogenesis of an epithelium / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / lung development / HDR through Homologous Recombination (HRR) / regulation of circadian rhythm / Dual Incision in GG-NER / DNA-templated DNA replication / Orc1 removal from chromatin / Dual incision in TC-NER / G1/S transition of mitotic cell cycle / Gap-filling DNA repair synthesis and ligation in TC-NER / circadian rhythm / nucleosome assembly / cellular response to xenobiotic stimulus / site of double-strand break / mitotic cell cycle / single-stranded DNA binding / Processing of DNA double-strand break ends / 4 iron, 4 sulfur cluster binding / histone binding / peptidyl-serine phosphorylation / DNA replication / DNA helicase / cell population proliferation / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nuclear body / Ub-specific processing proteases / cell cycle / cell division / intracellular membrane-bounded organelle
Similarity search - Function
DNA polymerase epsilon subunit B, N-terminal / DNA polymerases epsilon N terminal / Claspin / Timeless, C-terminal / Timeless PAB domain / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless / Timeless, N-terminal ...DNA polymerase epsilon subunit B, N-terminal / DNA polymerases epsilon N terminal / Claspin / Timeless, C-terminal / Timeless PAB domain / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless / Timeless, N-terminal / Timeless protein / DNA polymerase epsilon, subunit B / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain 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 / MCM4, winged helix domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA replication licensing factor Mcm5 / DNA polymerase alpha/epsilon subunit B / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / 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 / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 4 WD40 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 polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Cell division control protein 45 homolog / WD repeat and HMG-box DNA-binding protein 1 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / DNA polymerase epsilon subunit 2 / DNA polymerase epsilon catalytic subunit A / DNA replication licensing factor MCM6 ...Cell division control protein 45 homolog / WD repeat and HMG-box DNA-binding protein 1 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / DNA polymerase epsilon subunit 2 / DNA polymerase epsilon catalytic subunit A / 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 speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsJones MJ / Yeeles JTP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) United Kingdom
CitationJournal: EMBO J / Year: 2021
Title: Structure of a human replisome shows the organisation and interactions of a DNA replication machine.
Authors: Morgan L Jones / Yasemin Baris / Martin R G Taylor / Joseph T P Yeeles /
Abstract: The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45-MCM-GINS (CMG) helicase, which, in addition to ...The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45-MCM-GINS (CMG) helicase, which, in addition to unwinding the parental DNA duplex, arranges many proteins including the leading-strand polymerase Pol ε, together with TIMELESS-TIPIN, CLASPIN and AND-1 that have key and varied roles in maintaining smooth replisome progression. How these proteins are coordinated in the human replisome is poorly understood. We have determined a 3.2 Å cryo-EM structure of a human replisome comprising CMG, Pol ε, TIMELESS-TIPIN, CLASPIN and AND-1 bound to replication fork DNA. The structure permits a detailed understanding of how AND-1, TIMELESS-TIPIN and Pol ε engage CMG, reveals how CLASPIN binds to multiple replisome components and identifies the position of the Pol ε catalytic domain. Furthermore, the intricate network of contacts contributed by MCM subunits and TIMELESS-TIPIN with replication fork DNA suggests a mechanism for strand separation.
History
DepositionAug 24, 2021-
Header (metadata) releaseNov 10, 2021-
Map releaseNov 10, 2021-
UpdateDec 22, 2021-
Current statusDec 22, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.278
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.278
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13457.png

Downloads & links

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Map

FileDownload / File: emd_13457.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.145 Å
Density
Contour LevelBy AUTHOR: 0.278 / Movie #1: 0.278
Minimum - Maximum-0.3821279 - 1.6449642
Average (Standard dev.)0.006670034 (±0.06161397)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 458.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1451.1451.145
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z458.000458.000458.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.3821.6450.007

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Supplemental data

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Mask #1

Fileemd_13457_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened consensus cryoSPARC refinement lagging strand density

Fileemd_13457_additional_1.map
AnnotationSharpened consensus cryoSPARC refinement lagging strand density
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13457_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13457_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Core human replisome

EntireName: Core human replisome
Components
  • Complex: Core human replisome

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Supramolecule #1: Core human replisome

SupramoleculeName: Core human replisome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#19
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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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: 7.8
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 39.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.1 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 490000
CTF correctionSoftware - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 110000
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION

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Atomic model buiding 1

RefinementSpace: REAL

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