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- PDB-8thd: Structure of the Saccharomyces cerevisiae clamp unloader Elg1-RFC... -

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Basic information

Entry
Database: PDB / ID: 8thd
TitleStructure of the Saccharomyces cerevisiae clamp unloader Elg1-RFC bound to PCNA
Components
  • (Replication factor C subunit ...) x 4
  • ELG1 isoform 1
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / DNA replication / DNA sliding clamp / PCNA clamp / clamp loader/unloader / Elg1-RFC unloader
Function / homology
Function and homology information


Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / Polymerase switching / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Translesion Synthesis by POLH ...Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / Polymerase switching / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Translesion Synthesis by POLH / DNA replication checkpoint signaling / PCNA complex / Termination of translesion DNA synthesis / Activation of ATR in response to replication stress / lagging strand elongation / sister chromatid cohesion / mitotic sister chromatid cohesion / error-free translesion synthesis / DNA polymerase processivity factor activity / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / regulation of DNA replication / Dual incision in TC-NER / mismatch repair / DNA damage checkpoint signaling / DNA-templated DNA replication / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
RFC1-like, AAA+ ATPase lid domain / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Proliferating cell nuclear antigen signature 2. ...RFC1-like, AAA+ ATPase lid domain / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Proliferating cell nuclear antigen signature 2. / 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 / : / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Proliferating cell nuclear antigen / ELG1 isoform 1 / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 4 / Replication factor C subunit 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsZheng, F. / Yao, Y.N. / Georgescu, R. / O'Donnell, M.E. / Li, H.
Funding support United States, 3items
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) United States
CitationJournal: Sci Adv / Year: 2024
Title: Structure of the PCNA unloader Elg1-RFC.
Authors: Fengwei Zheng / Nina Y Yao / Roxana E Georgescu / Huilin Li / Michael E O'Donnell /
Abstract: During DNA replication, the proliferating cell nuclear antigen (PCNA) clamps are loaded onto primed sites for each Okazaki fragment synthesis by the AAA heteropentamer replication factor C (RFC). ...During DNA replication, the proliferating cell nuclear antigen (PCNA) clamps are loaded onto primed sites for each Okazaki fragment synthesis by the AAA heteropentamer replication factor C (RFC). PCNA encircling duplex DNA is quite stable and is removed from DNA by the dedicated clamp unloader Elg1-RFC. Here, we show the cryo-EM structure of Elg1-RFC in various states with PCNA. The structures reveal essential features of Elg1-RFC that explain how it is dedicated to PCNA unloading. Specifically, Elg1 contains two external loops that block opening of the Elg1-RFC complex for DNA binding, and an "Elg1 plug" domain that fills the central DNA binding chamber, thereby reinforcing the exclusive PCNA unloading activity of Elg1-RFC. Elg1-RFC was capable of unloading PCNA using non-hydrolyzable AMP-PNP. Both RFC and Elg1-RFC could remove PCNA from covalently closed circular DNA, indicating that PCNA unloading occurs by a mechanism that is distinct from PCNA loading. Implications for the PCNA unloading mechanism are discussed.
History
DepositionJul 14, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 2024Provider: repository / Type: Initial release
Revision 1.0May 22, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1May 29, 2024Group: Database references / Category: citation / citation_author
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Revision 1.2Nov 13, 2024Group: Data collection / Source and taxonomy / Structure summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ELG1 isoform 1
B: Replication factor C subunit 4
C: Replication factor C subunit 3
D: Replication factor C subunit 2
E: Replication factor C subunit 5
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
H: Proliferating cell nuclear antigen
hetero molecules


Theoretical massNumber of molelcules
Total (without water)335,12116
Polymers332,5288
Non-polymers2,5938
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 4 molecules AFGH

#1: Protein ELG1 isoform 1


Mass: 91391.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ELG1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4F7G7
#6: Protein Proliferating cell nuclear antigen


Mass: 29102.203 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PCNA / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6B7JGY6

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Replication factor C subunit ... , 4 types, 4 molecules BCDE

#2: Protein Replication factor C subunit 4 / Replication factor C4 / Activator 1 37 kDa subunit


Mass: 36201.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC4, YOL094C, O0923 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40339
#3: Protein Replication factor C subunit 3 / Replication factor C3 / Activator 1 40 kDa subunit


Mass: 37841.051 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC3, YNL290W, N0533 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38629
#4: Protein Replication factor C subunit 2 / Replication factor C2 / Activator 1 41 kDa subunit


Mass: 39794.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC2, YJR068W, J1808 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40348
#5: Protein Replication factor C subunit 5 / Replication factor C5 / Activator 1 40 kDa subunit


Mass: 39993.582 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC5, YBR087W, YBR0810 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38251

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Non-polymers , 3 types, 8 molecules

#7: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#9: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestN
Has protein modificationN

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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

Component
IDNameTypeEntity IDParent-IDSource
1PCNA clamp unloader Elg1-RFCCOMPLEX#1-#60MULTIPLE SOURCES
2PCNA clamp unloader Elg1-RFCCOMPLEX#1-#51RECOMBINANT
3PCNA clampCOMPLEX#61RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
23Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 1800 nm / Nominal defocus min: 1100 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173929 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00820723
ELECTRON MICROSCOPYf_angle_d0.8828038
ELECTRON MICROSCOPYf_dihedral_angle_d5.42770
ELECTRON MICROSCOPYf_chiral_restr0.0413291
ELECTRON MICROSCOPYf_plane_restr0.0063551

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