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- EMDB-0287: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-0287
TitleCryo-EM structure of S. cerevisiae Polymerase epsilon deltacat mutant
Map dataPol epsilon deltacat
Sample
  • Complex: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C-Pol2+C-Dpb2)
    • Protein or peptide: DNA polymerase epsilon subunit B
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
  • Ligand: ZINC ION
Function / homology
Function and homology information


DNA-templated DNA replication maintenance of fidelity / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / SUMO binding / DNA replication proofreading / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / mitotic DNA replication checkpoint signaling ...DNA-templated DNA replication maintenance of fidelity / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / SUMO binding / 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 / error-prone translesion synthesis / base-excision repair, gap-filling / replication fork / 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-directed DNA polymerase / DNA-directed DNA polymerase activity / cell cycle / nucleotide binding / mRNA binding / DNA binding / zinc ion binding / nucleus / cytoplasm
Similarity search - Function
DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B / DNA polymerase family B, exonuclease domain ...DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / 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 / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase epsilon catalytic subunit A / DNA polymerase epsilon subunit B
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsGoswami P / Nans A / Costa A
CitationJournal: Nat Commun / Year: 2018
Title: Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome.
Authors: Panchali Goswami / Ferdos Abid Ali / Max E Douglas / Julia Locke / Andrew Purkiss / Agnieszka Janska / Patrik Eickhoff / Anne Early / Andrea Nans / Alan M C Cheung / John F X Diffley / Alessandro Costa /
Abstract: Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, ...Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.
History
DepositionOct 10, 2018-
Header (metadata) releaseDec 12, 2018-
Map releaseDec 12, 2018-
UpdateDec 12, 2018-
Current statusDec 12, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0576
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0576
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hv8
  • Surface level: 0.0576
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0287.png

Downloads & links

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Map

FileDownload / File: emd_0287.map.gz / Format: CCP4 / Size: 23.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPol epsilon deltacat
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.0576 / Movie #1: 0.0576
Minimum - Maximum-0.1576485 - 0.29041803
Average (Standard dev.)0.0007833317 (±0.011617362)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions184184184
Spacing184184184
CellA=B=C: 200.56001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z184184184
origin x/y/z0.0000.0000.000
length x/y/z200.560200.560200.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS184184184
D min/max/mean-0.1580.2900.001

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C...

EntireName: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C-Pol2+C-Dpb2)
Components
  • Complex: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C-Pol2+C-Dpb2)
    • Protein or peptide: DNA polymerase epsilon subunit B
    • Protein or peptide: DNA polymerase epsilon catalytic subunit A
  • Ligand: ZINC ION

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Supramolecule #1: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C...

SupramoleculeName: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C-Pol2+C-Dpb2)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: DNA polymerase epsilon subunit B

MacromoleculeName: DNA polymerase epsilon subunit B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 78.408758 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MFGSGNVLPV KIQPPLLRPL AYRVLSRKYG LSIKSDGLSA LAEFVGTNIG ANWRQGPATI KFLEQFAAVW KQQERGLFID QSGVKEVIQ EMKEREKVEW SHEHPIQHEE NILGRTDDDE NNSDDEMPIA ADSSLQNVSL SSPMRQPTER DEYKQPFKPE S SKALDWRD ...String:
MFGSGNVLPV KIQPPLLRPL AYRVLSRKYG LSIKSDGLSA LAEFVGTNIG ANWRQGPATI KFLEQFAAVW KQQERGLFID QSGVKEVIQ EMKEREKVEW SHEHPIQHEE NILGRTDDDE NNSDDEMPIA ADSSLQNVSL SSPMRQPTER DEYKQPFKPE S SKALDWRD YFKVINASQQ QRFSYNPHKM QFIFVPNKKQ NGLGGIAGFL PDIEDKVQMF LTRYYLTNDR VMRNENFQNS DM FNPLSSM VSLQNELSNT NRQQQSSSNS ITPIKNLLGR DAQNFLLLGL LNKNFKGNWS LEDPSGSVEI DISQTIPTQG HYY VPGCMV LVEGIYYSVG NKFHVTSMTL PPGERREITL ETIGNLDLLG IHGISNNNFI ARLDKDLKIR LHLLEKELTD HKFV ILGAN LFLDDLKIMT ALSKILQKLN DDPPTLLIWQ GSFTSVPVFA SMSSRNISSS TQFKNNFDAL ATLLSRFDNL TENTT MIFI PGPNDLWGSM VSLGASGTLP QDPIPSAFTK KINKVCKNVV WSSNPTRIAY LSQEIVIFRD DLSGRFKRHR LEFPFN ESE DVYTENDNMM SKDTDIVPID ELVKEPDQLP QKVQETRKLV KTILDQGHLS PFLDSLRPIS WDLDHTLTLC PIPSTMV LC DTTSAQFDLT YNGCKVINPG SFIHNRRARY MEYVPSSKKT IQEEIYI

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Macromolecule #2: DNA polymerase epsilon catalytic subunit A

MacromoleculeName: DNA polymerase epsilon catalytic subunit A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 104.984844 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: SMIRKQAESY ANSTWEVLQY KDSGEPGVLE VFVTINGKVQ NITFHIPKTI YMKFKSQTMP LQKIKNCLIE KSSASLPNNP KTSNPAGGQ LFKITLPESV FLEEKENCTS IFNDENVLGV FEGTITPHQR AIMDLGASVT FRSKAMGALG KGIQQGFEMK D LSMAENER ...String:
SMIRKQAESY ANSTWEVLQY KDSGEPGVLE VFVTINGKVQ NITFHIPKTI YMKFKSQTMP LQKIKNCLIE KSSASLPNNP KTSNPAGGQ LFKITLPESV FLEEKENCTS IFNDENVLGV FEGTITPHQR AIMDLGASVT FRSKAMGALG KGIQQGFEMK D LSMAENER YLSGFSMDIG YLLHFPTSIG YEFFSLFKSW GDTITILVLK PSNQAQEINA SSLGQIYKQM FEKKKGKIET YS YLVDIKE DINFEFVYFT DISKLYRRLS QETTKLKEER GLQFLLLLQS PFITKLLGTI RLLNQMPIVK LSLNEVLLPQ LNW QPTLLK KLVNHVLSSG SWISHLIKLS QYSNIPICNL RLDSMDYIID VLYARKLKKE NIVLWWNEKA PLPDHGGIQN DFDL NTSWI MNDSEFPKIN NSGVYDNVVL DVGVDNLTVN TILTSALIND AEGSDLVNNN MGIDDKDAVI NSPSEFVHDA FSNDA LNVL RGMLKEWWDE ALKENSTADL LVNSLASWVQ NPNAKLFDGL LRYHVHNLTK KALLQLVNEF SALGSTIVYA DRNQIL IKT NKYSPENCYA YSQYMMKAVR TNPMFSYLDL NIKRYWDLLI WMDKFNFSGL ACIEIEEKEN QDYTAVSQWQ LKKFLSP IY QPEFEDWMMI ILDSMLKTKQ SYLKLNSGTQ RPTQIVNVKK QDKEDSVENS LNGFSHLFSK PLMKRVKKLF KNQQEFIL D PQYEADYVIP VLPGSHLNVK NPLLELVKSL CHVMLLSKST ILEIRTLRKE LLKIFELREF AKVAEFKDPS LSLVVPDFL CEYCFFISDI DFCKAAPESI FSCVRCHKAF NQVLLQEHLI QKLRSDIESY LIQDLRCSRC HKVKRDYMSA HCPCAGAWEG TLPRESIVQ KLNVFKQVAK YYGFDILLSC IADLT

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 161376
DetailsVolta phase plate

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