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- EMDB-0287: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat mutant -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-0287 | |||||||||
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Title | Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat mutant | |||||||||
![]() | Pol epsilon deltacat | |||||||||
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Function / homology | ![]() DNA-templated DNA replication maintenance of fidelity / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / SUMO binding / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / Termination of translesion DNA synthesis / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling ...DNA-templated DNA replication maintenance of fidelity / gene conversion / DNA replication initiation / epsilon DNA polymerase complex / SUMO binding / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / Termination of translesion DNA synthesis / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / nucleotide-excision repair, DNA gap filling / mitotic sister chromatid cohesion / DNA replication proofreading / leading strand elongation / nuclear replication fork / Dual incision in TC-NER / 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 / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / mRNA binding / DNA binding / zinc ion binding / nucleus / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.4 Å | |||||||||
![]() | Goswami P / Nans A / Costa A | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 15.7 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.3 KB 12.3 KB | Display Display | ![]() |
Images | ![]() | 121.3 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6hv8MC ![]() 0288C ![]() 6hv9C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Pol epsilon deltacat | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.09 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C...
Entire | Name: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C-Pol2+C-Dpb2) |
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Components |
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-Supramolecule #1: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C...
Supramolecule | Name: Cryo-EM structure of S. cerevisiae Polymerase epsilon deltacat (C-Pol2+C-Dpb2) type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #1: DNA polymerase epsilon subunit B
Macromolecule | Name: DNA polymerase epsilon subunit B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 78.408758 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: DNA polymerase epsilon catalytic subunit A
Macromolecule | Name: DNA polymerase epsilon catalytic subunit A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 104.984844 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #3: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.6 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Details | Volta phase plate |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 161376 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |