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- EMDB-24625: Archaeal DNA ligase and heterotrimeric PCNA in complex with end-j... -

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Basic information

Entry
Database: EMDB / ID: EMD-24625
TitleArchaeal DNA ligase and heterotrimeric PCNA in complex with end-joined DNA
Map dataSharpened map
Sample
  • Complex: Ternary complex of DNA Ligase with PCNA1-2-3 and end-joined DNA
    • Protein or peptide: DNA polymerase sliding clamp 1
    • Protein or peptide: DNA polymerase sliding clamp 2
    • Protein or peptide: DNA polymerase sliding clamp 3
    • DNA: End-joined DNA
    • DNA: Template strand DNA
    • Protein or peptide: DNA ligase
  • Ligand: MANGANESE (II) ION
KeywordsDNA ligase / PCNA / cryo-EM / LIGASE-DNA complex
Function / homology
Function and homology information


DNA ligase (ATP) / DNA ligase (ATP) activity / lagging strand elongation / DNA biosynthetic process / DNA polymerase processivity factor activity / leading strand elongation / regulation of DNA replication / DNA recombination / cell division / DNA repair ...DNA ligase (ATP) / DNA ligase (ATP) activity / lagging strand elongation / DNA biosynthetic process / DNA polymerase processivity factor activity / leading strand elongation / regulation of DNA replication / DNA recombination / cell division / DNA repair / DNA binding / ATP binding / metal ion binding
Similarity search - Function
DNA ligase, ATP-dependent, bacterial/archaeal / : / DNA ligase, ATP-dependent / DNA ligase, ATP-dependent, N-terminal / DNA ligase, ATP-dependent, N-terminal domain superfamily / DNA ligase N terminus / ATP-dependent DNA ligase AMP-binding site. / ATP-dependent DNA ligase signature 2. / DNA ligase, ATP-dependent, C-terminal / ATP dependent DNA ligase C terminal region ...DNA ligase, ATP-dependent, bacterial/archaeal / : / DNA ligase, ATP-dependent / DNA ligase, ATP-dependent, N-terminal / DNA ligase, ATP-dependent, N-terminal domain superfamily / DNA ligase N terminus / ATP-dependent DNA ligase AMP-binding site. / ATP-dependent DNA ligase signature 2. / DNA ligase, ATP-dependent, C-terminal / ATP dependent DNA ligase C terminal region / DNA ligase, ATP-dependent, conserved site / ATP-dependent DNA ligase family profile. / DNA ligase, ATP-dependent, central / ATP dependent DNA ligase domain / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / : / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA polymerase sliding clamp 3 / DNA polymerase sliding clamp 1 / DNA polymerase sliding clamp 2 / DNA ligase
Similarity search - Component
Biological speciesSaccharolobus solfataricus (archaea) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsSverzhinsky A / Pascal JM
Funding support United States, 2 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2015-05776 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA92584 United States
CitationJournal: Structure / Year: 2022
Title: Cryo-EM structures and biochemical insights into heterotrimeric PCNA regulation of DNA ligase.
Authors: Aleksandr Sverzhinsky / Alan E Tomkinson / John M Pascal /
Abstract: DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there are limited insights into the ...DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there are limited insights into the physical basis for this regulation. Here, we use single-particle cryoelectron microscopy (cryo-EM) to analyze an archaeal DNA ligase and heterotrimeric PCNA in complex with a single-strand DNA break. The cryo-EM structures highlight a continuous DNA-binding surface formed between DNA ligase and PCNA that supports the distorted conformation of the DNA break undergoing repair and contributes to PCNA stimulation of DNA ligation. DNA ligase is conformationally flexible within the complex, with its domains fully ordered only when encircling the repaired DNA to form a stacked ring structure with PCNA. The structures highlight DNA ligase structural transitions while docked on PCNA, changes in DNA conformation during ligation, and the potential for DNA ligase domains to regulate PCNA accessibility to other repair factors.
History
DepositionAug 4, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0691
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0691
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rpx
  • Surface level: 0.0691
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24625.png

Downloads & links

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Map

FileDownload / File: emd_24625.map.gz / Format: CCP4 / Size: 24.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.5 Å/pix.
x 186 pix.
= 279. Å		1.5 Å/pix.
x 186 pix.
= 279. Å		1.5 Å/pix.
x 186 pix.
= 279. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0691 / Movie #1: 0.0691
Minimum - Maximum-0.025438115 - 1.8002273
Average (Standard dev.)0.0015828998 (±0.02249158)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions186186186
Spacing186186186
CellA=B=C: 279.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.51.51.5
M x/y/z186186186
origin x/y/z0.0000.0000.000
length x/y/z279.000279.000279.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS186186186
D min/max/mean-0.0251.8000.002

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Supplemental data

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Additional map: Unsharpened map

Fileemd_24625_additional_1.map
AnnotationUnsharpened 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 : Ternary complex of DNA Ligase with PCNA1-2-3 and end-joined DNA

EntireName: Ternary complex of DNA Ligase with PCNA1-2-3 and end-joined DNA
Components
  • Complex: Ternary complex of DNA Ligase with PCNA1-2-3 and end-joined DNA
    • Protein or peptide: DNA polymerase sliding clamp 1
    • Protein or peptide: DNA polymerase sliding clamp 2
    • Protein or peptide: DNA polymerase sliding clamp 3
    • DNA: End-joined DNA
    • DNA: Template strand DNA
    • Protein or peptide: DNA ligase
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Ternary complex of DNA Ligase with PCNA1-2-3 and end-joined DNA

SupramoleculeName: Ternary complex of DNA Ligase with PCNA1-2-3 and end-joined DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Saccharolobus solfataricus (archaea)
Molecular weightTheoretical: 175 KDa

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Macromolecule #1: DNA polymerase sliding clamp 1

MacromoleculeName: DNA polymerase sliding clamp 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharolobus solfataricus (archaea) / Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2
Molecular weightTheoretical: 28.711961 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAFKIVYPNA KDFFSFINSI TNVTDSIILN FTEDGIFSRH LTEDKVLMAI MRIPKDVLSE YSIDSPTSVK LDVSSVKKIL SKASSKKAT IELTETDSGL KIIIRDEKSG AKSTIYIKAE KGQVEQLTEP KVNLAVNFTT DESVLNVIAA DVTLVGEEMR I STEEDKIK ...String:
MAFKIVYPNA KDFFSFINSI TNVTDSIILN FTEDGIFSRH LTEDKVLMAI MRIPKDVLSE YSIDSPTSVK LDVSSVKKIL SKASSKKAT IELTETDSGL KIIIRDEKSG AKSTIYIKAE KGQVEQLTEP KVNLAVNFTT DESVLNVIAA DVTLVGEEMR I STEEDKIK IEAGEEGKRY VAFLMKDKPL KELSIDTSAS SSYSAEMFKD AVKGLRGFSA PTMVSFGENL PMKIDVEAVS GG HMIFWIA PRLLEHHHHH H

UniProtKB: DNA polymerase sliding clamp 1

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Macromolecule #2: DNA polymerase sliding clamp 2

MacromoleculeName: DNA polymerase sliding clamp 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharolobus solfataricus (archaea) / Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2
Molecular weightTheoretical: 27.461084 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKAKVIDAVS FSYILRTVGD FLSEANFIVT KEGIRVSGID PSRVVFLDIF LPSSYFEGFE VSQEKEIIGF KLEDVNDILK RVLKDDTLI LSSNESKLTL TFDGEFTRSF ELPLIQVEST QPPSVNLEFP FKAQLLTITF ADIIDELSDL GEVLNIHSKE N KLYFEVIG ...String:
MKAKVIDAVS FSYILRTVGD FLSEANFIVT KEGIRVSGID PSRVVFLDIF LPSSYFEGFE VSQEKEIIGF KLEDVNDILK RVLKDDTLI LSSNESKLTL TFDGEFTRSF ELPLIQVEST QPPSVNLEFP FKAQLLTITF ADIIDELSDL GEVLNIHSKE N KLYFEVIG DLSTAKVELS TDNGTLLEAS GADVSSSYGM EYVANTTKMR RASDSMELYF GSQIPLKLRF KLPQEGYGDF YI APRAD

UniProtKB: DNA polymerase sliding clamp 2

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Macromolecule #3: DNA polymerase sliding clamp 3

MacromoleculeName: DNA polymerase sliding clamp 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharolobus solfataricus (archaea) / Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2
Molecular weightTheoretical: 28.560268 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKVVYDDVRV LKDIIQALAR LVDEAVLKFK QDSVELVALD RAHISLISVN LPREMFKEYD VNDEFKFGFN TQYLMKILKV AKRKEAIEI ASESPDSVII NIIGSTNREF NVRNLEVSEQ EIPEINLQFD ISATISSDGF KSAISEVSTV TDNVVVEGHE D RILIKAEG ...String:
MKVVYDDVRV LKDIIQALAR LVDEAVLKFK QDSVELVALD RAHISLISVN LPREMFKEYD VNDEFKFGFN TQYLMKILKV AKRKEAIEI ASESPDSVII NIIGSTNREF NVRNLEVSEQ EIPEINLQFD ISATISSDGF KSAISEVSTV TDNVVVEGHE D RILIKAEG ESEVEVEFSK DTGGLQDLEF SKESKNSYSA EYLDDVLSLT KLSDYVKISF GNQKPLQLFF NMEGGGKVTY LL APKVLEH HHHHH

UniProtKB: DNA polymerase sliding clamp 3

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Macromolecule #6: DNA ligase

MacromoleculeName: DNA ligase / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA ligase (ATP)
Source (natural)Organism: Saccharolobus solfataricus (archaea) / Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2
Molecular weightTheoretical: 69.998375 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MEFKVIAEYF DKLEKISSRL QLTALLADLL SKSDKTIIDK VVYIIQGKLW PDFLGYPELG IGEKFLIKA ISIATNTDEN SVENLYKTIG DLGEVARRLK SKQQSTGILG FLGTTSKESL TVDEVYSTLS KVALTTGEGS R DLKIRLLA ...String:
MGSSHHHHHH SSGLVPRGSH MEFKVIAEYF DKLEKISSRL QLTALLADLL SKSDKTIIDK VVYIIQGKLW PDFLGYPELG IGEKFLIKA ISIATNTDEN SVENLYKTIG DLGEVARRLK SKQQSTGILG FLGTTSKESL TVDEVYSTLS KVALTTGEGS R DLKIRLLA GLLKKADPLE AKFLVRFVEG RLRVGIGDAT VLDAMAIAFG GGQSASEIIE RAYNLRADLG NIAKIIVEKG IE ALKTLKP QVGIPIRPML AERLSNPEEI LKKMGGNAIV DYKYDGERAQ IHKKEDKIFI FSRRLENITS QYPDVVDYVS KYI EGKEFI IEGEIVAIDP ESGEMRPFQE LMHRKRKSDI YEAIKEYPVN VFLFDLMYYE DVDYTTKPLE ARRKLLESIV KPND YVKIA HHIQANNVED LKSFFYRAIS EGGEGVMVKA IGKDAIYQAG ARGWLWIKLK RDYQSEMADT VDLVVVGGFY GKGKR GGKI SSLLMAAYNP KTDSFESVCK VASGFSDEQL DELQKKLMEI KRDVKHPRVN SKMEPDIWVE PVYVAEIIGS EITISP LHT CCQDVVEKDA GLSIRFPRFI RWRDDKSPED ATTTDEILEM YNKQPKKKIE SPAVDESV

UniProtKB: DNA ligase

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Macromolecule #4: End-joined DNA

MacromoleculeName: End-joined DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.549299 KDa
SequenceString:
(DG)(DT)(DA)(DT)(DC)(DC)(DT)(DC)(DG)(DT) (DA)(DG)(DT)(DG)(DC)(DA)(DG)(DA)(DT)(DG) (DC)(DG)(DT)(DC)(DG)(DT)(DC)(DG)(DG) (DA)(DC)(DT)(DG)(DA)(DT)(DT)(DC)(DG)(DG) (DT) (DA)(DG)(DA)(DT)(DC)(DT)(DG)

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Macromolecule #5: Template strand DNA

MacromoleculeName: Template strand DNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.403264 KDa
SequenceString:
(DC)(DA)(DG)(DA)(DT)(DC)(DT)(DA)(DC)(DC) (DG)(DA)(DA)(DT)(DC)(DA)(DG)(DT)(DC)(DC) (DG)(DA)(DC)(DG)(DA)(DC)(DG)(DC)(DA) (DT)(DC)(DT)(DG)(DC)(DA)(DC)(DT)(DA)(DC) (DG) (DA)(DG)(DG)(DA)(DT)(DA)(DC)

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Macromolecule #7: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 7 / Number of copies: 3 / Formula: MN
Molecular weightTheoretical: 54.938 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

Concentration0.35 mg/mL
BufferpH: 7.5
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: wait time 0, blot force 1, blot time 1, drain time 0.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 8060 / Average electron dose: 100.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 192490
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)

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Atomic model buiding 1

Initial model
PDB IDChain

2hix

source_name: PDB, initial_model_type: experimental model

2hii

source_name: PDB, initial_model_type: experimental model

1x9n

chain_id: B, source_name: PDB, initial_model_type: experimental model

1x9n

chain_id: C, source_name: PDB, initial_model_type: experimental model

1x9n

chain_id: D, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7rpx:
Archaeal DNA ligase and heterotrimeric PCNA in complex with end-joined DNA

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