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- EMDB-44356: Cryo-EM structure of S. cerevisiae PolE-core-DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-44356
TitleCryo-EM structure of S. cerevisiae PolE-core-DNA
Map datastructure of S. cerevisiae PolE-core-DNA
Sample
  • Complex: PolE-core-DNA complex
    • DNA: Primer DNA
    • DNA: Template DNA
  • Protein or peptide: DNA polymerase epsilon catalytic subunit
  • Ligand: IRON/SULFUR CLUSTER
KeywordsPol2 / DNA / REPLICATION / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


epsilon DNA polymerase complex / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / nucleotide binding / DNA binding / zinc ion binding
Similarity search - Function
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-directed DNA polymerase, family B, multifunctional domain / 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 epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / 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 / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase epsilon catalytic subunit
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / DNA molecule (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsYuan Z / Georgescu R / O'Donnell M / Li H
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115809 United States
Howard Hughes Medical Institute (HHMI)M.E.O. United States
CitationJournal: Science / Year: 2024
Title: Mechanism of PCNA loading by Ctf18-RFC for leading-strand DNA synthesis.
Authors: Zuanning Yuan / Roxana Georgescu / Nina Y Yao / Olga Yurieva / Michael E O'Donnell / Huilin Li /
Abstract: The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific ...The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific to the leading-strand Pol (Polε). We reveal here the underlying mechanism of Ctf18-RFC specificity to Polε using cryo-electron microscopy and biochemical studies. We found that both Ctf18-RFC and Polε contain specific structural features that direct PCNA loading onto DNA. Unlike other clamp loaders, Ctf18-RFC has a disordered ATPase associated with a diverse cellular activities (AAA+) motor that requires Polε to bind and stabilize it for efficient PCNA loading. In addition, Ctf18-RFC can pry prebound Polε off of DNA, then load PCNA onto DNA and transfer the PCNA-DNA back to Polε. These elements in both Ctf18-RFC and Polε provide specificity in loading PCNA onto DNA for Polε.
History
DepositionMar 31, 2024-
Header (metadata) releaseOct 9, 2024-
Map releaseOct 9, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44356.png

Downloads & links

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Map

FileDownload / File: emd_44356.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of S. cerevisiae PolE-core-DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.68114114 - 1.9891208
Average (Standard dev.)0.002016878 (±0.046768963)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half A map

Fileemd_44356_half_map_1.map
Annotationhalf A map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half B map

Fileemd_44356_half_map_2.map
AnnotationHalf B 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 : PolE-core-DNA complex

EntireName: PolE-core-DNA complex
Components
  • Complex: PolE-core-DNA complex
    • DNA: Primer DNA
    • DNA: Template DNA
  • Protein or peptide: DNA polymerase epsilon catalytic subunit
  • Ligand: IRON/SULFUR CLUSTER

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Supramolecule #1: PolE-core-DNA complex

SupramoleculeName: PolE-core-DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Primer DNA

MacromoleculeName: Primer DNA / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 3.026977 KDa
SequenceString:
(DC)(DT)(DG)(DT)(DT)(DG)(DC)(DT)(DG)(DC)

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Macromolecule #2: Template DNA

MacromoleculeName: Template DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 3.680432 KDa
SequenceString:
(DT)(DA)(DG)(DC)(DA)(DG)(DC)(DA)(DA)(DC) (DA)(DG)

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Macromolecule #3: DNA polymerase epsilon catalytic subunit

MacromoleculeName: DNA polymerase epsilon catalytic subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 133.898031 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: SNNYALSAQQ LLNASKIDDI DSMMGFERYV PPQYNGRFDA KDIDQIPGRV GWLTNMHATL VSQETLSSGS NGGGNSNDGE RVTTNQGIS GVDFYFLDEE GGSFKSTVVY DPYFFIACND ESRVNDVEEL VKKYLESCLK SLQIIRKEDL TMDNHLLGLQ K TLIKLSFV ...String:
SNNYALSAQQ LLNASKIDDI DSMMGFERYV PPQYNGRFDA KDIDQIPGRV GWLTNMHATL VSQETLSSGS NGGGNSNDGE RVTTNQGIS GVDFYFLDEE GGSFKSTVVY DPYFFIACND ESRVNDVEEL VKKYLESCLK SLQIIRKEDL TMDNHLLGLQ K TLIKLSFV NSNQLFEARK LLRPILQDNA NNNVQRNIYN VAANGSEKVD AKHLIEDIRE YDVPYHVRVS IDKDIRVGKW YK VTQQGFI EDTRKIAFAD PVVMAFDIET TKPPLKFPDS AVDQIMMISY MIDGEGFLIT NREIISEDIE DFEYTPKPEY PGF FTIFNE NDEVALLQRF FEHIRDVRPT VISTFNGDFF DWPFIHNRSK IHGLDMFDEI GFAPDAEGEY KSSYCSHMDC FRWV KRDSY LPQGSQGLKA VTQSKLGYNP IELDPELMTP YAFEKPQHLS EYSVSDAVAT YYLYMKYVHP FIFSLCTIIP LNPDE TLRK GTGTLCEMLL MVQAYQHNIL LPNKHTDPIE RFYDGHLLES ETYVGGHVES LEAGVFRSDL KNEFKIDPSA IDELLQ ELP EALKFSVEVE NKSSVDKVTN FEEIKNQITQ KLLELKENNI RNELPLIYHV DVASMYPNIM TTNRLQPDSI KAERDCA SC DFNRPGKTCA RKLKWAWRGE FFPSKMDEYN MIKRALQNET FPNKNKFSKK KVLTFDELSY ADQVIHIKKR LTEYSRKV Y HRVKVSEIVE REAIVCQREN PFYVDTVKSF RDRRYEFKGL AKTWKGNLSK IDPSDKHARD EAKKMIVLYD SLQLAHKVI LNSFYGYVMR KGSRWYSMEM AGITCLTGAT IIQMARALVE RVGRPLELDT DGIWCILPKS FPETYFFTLE NGKKLYLSYP CSMLNYRVH QKFTNHQYQE LKDPLNYIYE THSENTIFFE VDGPYKAMIL PSSKEEGKGI KKRYAVFNED GSLAELKGFE L KRRGELQL IKNFQSDIFK VFLEGDTLEG CYSAVASVCN RWLDVLDSHG LMLEDEDLVS LICENRSMSK TLKEYEGQKS TS ITTARRL GDFLGEDMVK DKGLQCKYII SSKPFNAPVT ERAIPVAIFS ADIPIKRSFL RRWTLDPSLE DLDIRTIIDW GYY RERLGS AIQKIITIPA ALQGVSNPVP RVEHPDWLKR KIAT

UniProtKB: DNA polymerase epsilon catalytic subunit

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Macromolecule #4: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 121833
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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