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- EMDB-10089: Cryo-EM structure of Ctf18-1-8 in complex with the catalytic doma... -

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Basic information

Entry
Database: EMDB / ID: EMD-10089
TitleCryo-EM structure of Ctf18-1-8 in complex with the catalytic domain of DNA polymerase epsilon (Class 2)
Map data
Sample
  • Complex: Complex of the catalytic domain of DNA polymerase epsilon with the Ctf18-1-8 module of Ctf18-RFC
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Protein or peptide: Chromosome transmission fidelity protein 8
    • Protein or peptide: Chromosome transmission fidelity protein 18
    • Protein or peptide: Sister chromatid cohesion protein DCC1
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
Function / homology
Function and homology information


maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / Ctf18 RFC-like complex / nucleotide-excision repair, DNA gap filling / SUMO binding / maintenance of DNA trinucleotide repeats / DNA replication proofreading / Activation of the pre-replicative complex ...maintenance of mitotic sister chromatid cohesion / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / Ctf18 RFC-like complex / nucleotide-excision repair, DNA gap filling / SUMO binding / maintenance of DNA trinucleotide repeats / DNA replication proofreading / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / mitotic sister chromatid cohesion / leading strand elongation / nuclear replication fork / chromosome, centromeric region / DNA replication initiation / error-prone translesion synthesis / base-excision repair, gap-filling / replication fork / double-strand break repair via homologous recombination / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / single-stranded DNA binding / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / mRNA binding / chromatin / ATP hydrolysis activity / mitochondrion / DNA binding / zinc ion binding / ATP binding / nucleus
Similarity search - Function
Chromosome transmission fidelity protein 8 / Ctf8 / Sister chromatid cohesion protein Dcc1 / Sister chromatid cohesion protein Dcc1 / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / : / DNA polymerase family B, thumb domain ...Chromosome transmission fidelity protein 8 / Ctf8 / Sister chromatid cohesion protein Dcc1 / Sister chromatid cohesion protein Dcc1 / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / : / DNA polymerase family B, thumb domain / 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 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ribonuclease H superfamily / Ribonuclease H-like superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA polymerase epsilon catalytic subunit A / Sister chromatid cohesion protein DCC1 / Chromosome transmission fidelity protein 8 / Chromosome transmission fidelity protein 18
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsGrabarczyk DB / Song B
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research FoundationGR 5152/3-1 Germany
CitationJournal: Nucleic Acids Res / Year: 2020
Title: Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication.
Authors: Katy Stokes / Alicja Winczura / Boyuan Song / Giacomo De Piccoli / Daniel B Grabarczyk /
Abstract: The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA ...The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.
History
DepositionJun 20, 2019-
Header (metadata) releaseJul 8, 2020-
Map releaseJul 8, 2020-
UpdateJan 20, 2021-
Current statusJan 20, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
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  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6s2f
  • Surface level: 0.05
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10089.png

Downloads & links

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Map

FileDownload / File: emd_10089.map.gz / Format: CCP4 / Size: 93 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0635 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.0649455 - 0.19043693
Average (Standard dev.)1.2489782e-05 (±0.006298232)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions290290290
Spacing290290290
CellA=B=C: 308.415 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06351.06351.0635
M x/y/z290290290
origin x/y/z0.0000.0000.000
length x/y/z308.415308.415308.415
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS290290290
D min/max/mean-0.0650.1900.000

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Supplemental data

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Half map: #1

Fileemd_10089_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10089_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of the catalytic domain of DNA polymerase epsilon with th...

EntireName: Complex of the catalytic domain of DNA polymerase epsilon with the Ctf18-1-8 module of Ctf18-RFC
Components
  • Complex: Complex of the catalytic domain of DNA polymerase epsilon with the Ctf18-1-8 module of Ctf18-RFC
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
    • Protein or peptide: Chromosome transmission fidelity protein 8
    • Protein or peptide: Chromosome transmission fidelity protein 18
    • Protein or peptide: Sister chromatid cohesion protein DCC1
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster

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Supramolecule #1: Complex of the catalytic domain of DNA polymerase epsilon with th...

SupramoleculeName: Complex of the catalytic domain of DNA polymerase epsilon with the Ctf18-1-8 module of Ctf18-RFC
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: DNA polymerase epsilon catalytic subunit A

MacromoleculeName: DNA polymerase epsilon catalytic subunit A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 140.39725 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKHHHHHHSA GLEVLFQGPG TGSEFELMMF GKKKNNGGSS TARYSAGNKY NTLSNNYALS AQQLLNASKI DDIDSMMGFE RYVPPQYNG RFDAKDIDQI PGRVGWLTNM HATLVSQETL SSGSNGGGNS NDGERVTTNQ GISGVDFYFL DEEGGSFKST V VYDPYFFI ...String:
MKHHHHHHSA GLEVLFQGPG TGSEFELMMF GKKKNNGGSS TARYSAGNKY NTLSNNYALS AQQLLNASKI DDIDSMMGFE RYVPPQYNG RFDAKDIDQI PGRVGWLTNM HATLVSQETL SSGSNGGGNS NDGERVTTNQ GISGVDFYFL DEEGGSFKST V VYDPYFFI ACNDESRVND VEELVKKYLE SCLKSLQIIR KEDLTMDNHL LGLQKTLIKL SFVNSNQLFE ARKLLRPILQ DN ANNNVQR NIYNVAANGS EKVDAKHLIE DIREYDVPYH VRVSIDKDIR VGKWYKVTQQ GFIEDTRKIA FADPVVMAFA IAT TKPPLK FPDSAVDQIM MISYMIDGEG FLITNREIIS EDIEDFEYTP KPEYPGFFTI FNENDEVALL QRFFEHIRDV RPTV ISTFN GDFFDWPFIH NRSKIHGLDM FDEIGFAPDA EGEYKSSYCS HMDCFRWVKR DSYLPQGSQG LKAVTQSKLG YNPIE LDPE LMTPYAFEKP QHLSEYSVSD AVATYYLYMK YVHPFIFSLC TIIPLNPDET LRKGTGTLCE MLLMVQAYQH NILLPN KHT DPIERFYDGH LLESETYVGG HVESLEAGVF RSDLKNEFKI DPSAIDELLQ ELPEALKFSV EVENKSSVDK VTNFEEI KN QITQKLLELK ENNIRNELPL IYHVDVASMY PNIMTTNRLQ PDSIKAERDC ASCDFNRPGK TCARKLKWAW RGEFFPSK M DEYNMIKRAL QNETFPNKNK FSKKKVLTFD ELSYADQVIH IKKRLTEYSR KVYHRVKVSE IVEREAIVCQ RENPFYVDT VKSFRDRRYE FKGLAKTWKG NLSKIDPSDK HARDEAKKMI VLYDSLQLAH KVILNSFYGY VMRKGSRWYS MEMAGITCLT GATIIQMAR ALVERVGRPL ELDTDGIWCI LPKSFPETYF FTLENGKKLY LSYPCSMLNY RVHQKFTNHQ YQELKDPLNY I YETHSENT IFFEVDGPYK AMILPSSKEE GKGIKKRYAV FNEDGSLAEL KGFELKRRGE LQLIKNFQSD IFKVFLEGDT LE GCYSAVA SVCNRWLDVL DSHGLMLEDE DLVSLICENR SMSKTLKEYE GQKSTSITTA RRLGDFLGED MVKDKGLQCK YII SSKPFN APVTERAIPV AIFSADIPIK RSFLRRWTLD PSLEDLDIRT IIDWGYYRER LGSAIQKIIT IPAALQGVSN PVPR VEHPD WLKRKIATKE DKFK

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Macromolecule #2: Chromosome transmission fidelity protein 8

MacromoleculeName: Chromosome transmission fidelity protein 8 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 15.189688 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPSVDIDASQ WQKLTQSREK QTTVITPLGM MMLEIQGELE LPKDFASLAR RDSPNEGRFS EQDGETLIRF GSLQIDGERA TLFVGKKQR LLGKVTKLDV PMGIMHFNSK DNKVELVDVM KYKVIFKDRP LPIM

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Macromolecule #3: Chromosome transmission fidelity protein 18

MacromoleculeName: Chromosome transmission fidelity protein 18 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 3.859329 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GAMGNQTVKI WVKYNEGFSN AVRKNVTWNN LWE

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Macromolecule #4: Sister chromatid cohesion protein DCC1

MacromoleculeName: Sister chromatid cohesion protein DCC1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 44.133785 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSINLHSAPE YDPSYKLIQL TPELLDIIQD PVQNHQLRFK SLDKDKSEVV LCSHDKTWVL KQRKHSNTVL LMREFVPEQP ITFDETLLF GLSKPYMDVV GFAKTESEFE TRETHGELNL NSVPIYNGEL DFSDKIMKRS STKVIGTLEE LLENSPCSAL E GISKWHKI ...String:
MSINLHSAPE YDPSYKLIQL TPELLDIIQD PVQNHQLRFK SLDKDKSEVV LCSHDKTWVL KQRKHSNTVL LMREFVPEQP ITFDETLLF GLSKPYMDVV GFAKTESEFE TRETHGELNL NSVPIYNGEL DFSDKIMKRS STKVIGTLEE LLENSPCSAL E GISKWHKI GGSVKDGVLC ILSQDFLFKA LHVLLMSAMA ESLDLQHLNV EDTHHAVGKD IEDEFNPYTR EIIETVLNKF AV QEQEAEN NTWRLRIPFI AQWYGIQALR KYVSGISMPI DEFLIKWKSL FPPFFPCDID IDMLRGYHFK PTDKTVQYIA KST LPMDPK ERFKVLFRLQ SQWDLEDIKP LIEELNSRGM KIDSFIMKYA RRKRLGKKTV VTSR

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Macromolecule #5: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

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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

Concentration0.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
20.0 mMHEPES
1.0 mMTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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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 / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.4 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average exposure time: 75.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER / Details: Ab initio 3D reconstruction using cisTEM
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0 beta)
Final 3D classificationNumber classes: 8 / Avg.num./class: 23594 / Software - Name: RELION (ver. 3.0 beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0 beta)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0 beta) / Number images used: 24967
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

4m8o

,

5okc

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-6s2f:
Cryo-EM structure of Ctf18-1-8 in complex with the catalytic domain of DNA polymerase epsilon (Class 2)

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