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

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Basic information

Entry
Database: PDB / ID: 7rpx
TitleArchaeal DNA ligase and heterotrimeric PCNA in complex with end-joined DNA
Components
  • (DNA polymerase sliding clamp ...) x 3
  • DNA ligase
  • End-joined DNA
  • Template strand DNA
KeywordsLIGASE/DNA / DNA ligase / PCNA / cryo-EM / LIGASE-DNA complex
Function / homology
Function and homology information


DNA ligase (ATP) / DNA ligase (ATP) activity / DNA ligation / lagging strand elongation / DNA biosynthetic process / leading strand elongation / DNA polymerase processivity factor activity / regulation of DNA replication / DNA recombination / cell division ...DNA ligase (ATP) / DNA ligase (ATP) activity / DNA ligation / lagging strand elongation / DNA biosynthetic process / leading strand elongation / DNA polymerase processivity factor activity / 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 signature 2. / ATP-dependent DNA ligase AMP-binding site. / 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 signature 2. / ATP-dependent DNA ligase AMP-binding site. / 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 / DNA (> 10) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsSverzhinsky, A. / Pascal, J.M.
Funding support United States, 2items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 12, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 16, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: DNA polymerase sliding clamp 1
B: DNA polymerase sliding clamp 2
C: DNA polymerase sliding clamp 3
X: End-joined DNA
Z: Template strand DNA
E: DNA ligase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,8499
Polymers183,6846
Non-polymers1653
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, All components co-elute stoichiometrically
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14420 Å2
ΔGint-104 kcal/mol
Surface area70840 Å2

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Components

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DNA polymerase sliding clamp ... , 3 types, 3 molecules ABC

#1: Protein DNA polymerase sliding clamp 1 / Proliferating cell nuclear antigen homolog 1 / PCNA1


Mass: 28711.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus (archaea) / Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: pcn1, pcnA-like, SSO0397, C41_008 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 / References: UniProt: P57766
#2: Protein DNA polymerase sliding clamp 2 / Proliferating cell nuclear antigen homolog 2 / PCNA2


Mass: 27461.084 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus (archaea) / Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: pcn2, pcnA-2, SSO1047 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 / References: UniProt: Q97Z84
#3: Protein DNA polymerase sliding clamp 3 / Proliferating cell nuclear antigen homolog 3 / PCNA3


Mass: 28560.268 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus (archaea) / Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: pcn3, pcnA-1, SSO0405, C41_016 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 / References: UniProt: P57765

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DNA chain , 2 types, 2 molecules XZ

#4: DNA chain End-joined DNA


Mass: 14549.299 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain Template strand DNA


Mass: 14403.264 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Protein / Non-polymers , 2 types, 4 molecules E

#6: Protein DNA ligase / Polydeoxyribonucleotide synthase [ATP]


Mass: 69998.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus (archaea) / Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: lig, SSO0189 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 / References: UniProt: Q980T8, DNA ligase (ATP)
#7: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mn

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of DNA Ligase with PCNA1-2-3 and end-joined DNA
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.175 MDa / Experimental value: YES
Source (natural)Organism: Saccharolobus solfataricus (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: wait time 0, blot force 1, blot time 1, drain time 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 100 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 8060

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC3.1CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.19model refinement
10cryoSPARC3.1initial Euler assignment
11cryoSPARC3.1final Euler assignment
13cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192490 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
12HIX

2hix

2HIX1
22HII

2hii

2HII2
31X9N

1x9n

B1X9N3
41X9N

1x9n

C1X9N3
51X9N

1x9n

D1X9N3
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 120.74 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312074
ELECTRON MICROSCOPYf_angle_d0.54616546
ELECTRON MICROSCOPYf_dihedral_angle_d15.7924658
ELECTRON MICROSCOPYf_chiral_restr0.0411895
ELECTRON MICROSCOPYf_plane_restr0.0031892

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