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- EMDB-41252: Cryo-EM map of the Saccharomyces cerevisiae PCNA clamp unloader E... -

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Basic information

Entry
Database: EMDB / ID: EMD-41252
TitleCryo-EM map of the Saccharomyces cerevisiae PCNA clamp unloader Elg1-RFC complex
Map dataCryo-EM density map of PCNA unloader Elg1-RFC alone complex
Sample
  • Complex: PCNA clamp unloader Elg1-RFC
    • Complex: Elg1 subunit of PCNA clamp unloader Elg1-RFC
      • Protein or peptide: ELG1 isoform 1
    • Complex: Subunits Rfc 2-5 of PCNA clamp unloader Elg1-RFC
      • Protein or peptide: Replication factor C subunit 4
      • Protein or peptide: Replication factor C subunit 3
      • Protein or peptide: Replication factor C subunit 2
      • Protein or peptide: Replication factor C subunit 5
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsDNA replication / DNA sliding clamp / PCNA clamp / clamp unloader / Elg1-RFC unloader / REPLICATION
Function / homology
Function and homology information


DNA clamp unloading / Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / Elg1 RFC-like complex / Ctf18 RFC-like complex / DNA replication factor C complex / Polymerase switching / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK ...DNA clamp unloading / Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / Elg1 RFC-like complex / Ctf18 RFC-like complex / DNA replication factor C complex / Polymerase switching / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / sister chromatid cohesion / mitotic sister chromatid cohesion / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / 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
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 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core ...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 / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZheng F / Yao YN / Georgescu R / O'Donnell ME / 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) 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, 2023-
Header (metadata) releaseMay 22, 2024-
Map releaseMay 22, 2024-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41252.png

Downloads & links

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Map

FileDownload / File: emd_41252.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM density map of PCNA unloader Elg1-RFC alone complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 420 pix.
= 347.76 Å		0.83 Å/pix.
x 420 pix.
= 347.76 Å		0.83 Å/pix.
x 420 pix.
= 347.76 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: 0.225
Minimum - Maximum-1.4091928 - 2.5652242
Average (Standard dev.)0.0001510807 (±0.045259185)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 347.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-EM density map of Elg-RFC alone complex half-A

Fileemd_41252_half_map_1.map
AnnotationCryo-EM density map of Elg-RFC alone complex_half-A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM density map of Elg-RFC alone complex half-B

Fileemd_41252_half_map_2.map
AnnotationCryo-EM density map of Elg-RFC alone complex_half-B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PCNA clamp unloader Elg1-RFC

EntireName: PCNA clamp unloader Elg1-RFC
Components
  • Complex: PCNA clamp unloader Elg1-RFC
    • Complex: Elg1 subunit of PCNA clamp unloader Elg1-RFC
      • Protein or peptide: ELG1 isoform 1
    • Complex: Subunits Rfc 2-5 of PCNA clamp unloader Elg1-RFC
      • Protein or peptide: Replication factor C subunit 4
      • Protein or peptide: Replication factor C subunit 3
      • Protein or peptide: Replication factor C subunit 2
      • Protein or peptide: Replication factor C subunit 5
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: PCNA clamp unloader Elg1-RFC

SupramoleculeName: PCNA clamp unloader Elg1-RFC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5

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Supramolecule #2: Elg1 subunit of PCNA clamp unloader Elg1-RFC

SupramoleculeName: Elg1 subunit of PCNA clamp unloader Elg1-RFC / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: Subunits Rfc 2-5 of PCNA clamp unloader Elg1-RFC

SupramoleculeName: Subunits Rfc 2-5 of PCNA clamp unloader Elg1-RFC / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#5
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: ELG1 isoform 1

MacromoleculeName: ELG1 isoform 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 91.391445 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MKRHVSLSDI LTGNKRKVRR QDALQITIDD ENDTESGTFD ARTAKHDDSS VIFLNHSVVK PIEAVSTNHK SAKEFLMTKR TKEKCDDDD DDLIVISDKS PKSETNCSKI ALSQEHEDDI SIISTSRIKS SLLNERASKI KNFLKHETTD TFKRLNSISK L NEIEPPLP ...String:
MKRHVSLSDI LTGNKRKVRR QDALQITIDD ENDTESGTFD ARTAKHDDSS VIFLNHSVVK PIEAVSTNHK SAKEFLMTKR TKEKCDDDD DDLIVISDKS PKSETNCSKI ALSQEHEDDI SIISTSRIKS SLLNERASKI KNFLKHETTD TFKRLNSISK L NEIEPPLP LHQSIFPVGD KELSDRSVDI PLPFRTIPPL NHNFLPSDYE SLKDKNSASC IPVRYQAPVL LGTNIKRNTT LT WPQLFKP VTLKQVLIEP KLKLRIKNWI ETSFHTLEKP TLRNRLLNRI NPNKQQGSGD ELANFIVPDL EEDENLRPDF YRN GEANSS LSEFVPLMIL HGNSIGKKTL IQTIMREIAG DDNSYQIYEV NSNMNRSKKD LLDILLDFTT THYVKDSSKR KSDY GLVLF NDVDVLFKEH DRGYWAMISK LCEFSRRPLV LTCKDLSLVP SELIALASEQ NSLFHTKKIS TSTVYAFLTK YLKSL EIEV CDDWLRDVVK QNNADIRKCL MHLQFWCVDT EADLISSKNR LPVLTSTLGS SVKDISQLTD LLSINDVIGQ ATLNRS MVR QEIDSTTMTP EKVNTFQDQN LDDEMKLKFD YVIDYKLHLN DPNRQPLLPF ELNIYQHIQE QLEARYSYVR EANHRLD NE YLVNRFKKMT ESTLNFLASR IPKYDHLQSA RRTRNSKKIS DILNQFKGIY NDETLNENAE IDLLSATTQQ IKAEINPF V FEIAKSDANV KNENKQIFEL HSENVSERRY KDLVYQLSQE GVLKNVWFNA DPSIVVRKWE HLHSGFSKNK

UniProtKB: ELG1 isoform 1

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Macromolecule #2: Replication factor C subunit 4

MacromoleculeName: Replication factor C subunit 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 36.201039 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSKTLSLQLP WVEKYRPQVL SDIVGNKETI DRLQQIAKDG NMPHMIISGM PGIGKTTSVH CLAHELLGRS YADGVLELNA SDDRGIDVV RNQIKHFAQK KLHLPPGKHK IVILDEADSM TAGAQQALRR TMELYSNSTR FAFACNQSNK IIEPLQSRCA I LRYSKLSD ...String:
MSKTLSLQLP WVEKYRPQVL SDIVGNKETI DRLQQIAKDG NMPHMIISGM PGIGKTTSVH CLAHELLGRS YADGVLELNA SDDRGIDVV RNQIKHFAQK KLHLPPGKHK IVILDEADSM TAGAQQALRR TMELYSNSTR FAFACNQSNK IIEPLQSRCA I LRYSKLSD EDVLKRLLQI IKLEDVKYTN DGLEAIIFTA EGDMRQAINN LQSTVAGHGL VNADNVFKIV DSPHPLIVKK ML LASNLED SIQILRTDLW KKGYSSIDIV TTSFRVTKNL AQVKESVRLE MIKEIGLTHM RILEGVGTYL QLASMLAKIH KLN NKA

UniProtKB: Replication factor C subunit 4

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Macromolecule #3: Replication factor C subunit 3

MacromoleculeName: Replication factor C subunit 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 37.841051 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSTSTEKRSK ENLPWVEKYR PETLDEVYGQ NEVITTVRKF VDEGKLPHLL FYGPPGTGKT STIVALAREI YGKNYSNMVL ELNASDDRG IDVVRNQIKD FASTRQIFSK GFKLIILDEA DAMTNAAQNA LRRVIERYTK NTRFCVLANY AHKLTPALLS R CTRFRFQP ...String:
MSTSTEKRSK ENLPWVEKYR PETLDEVYGQ NEVITTVRKF VDEGKLPHLL FYGPPGTGKT STIVALAREI YGKNYSNMVL ELNASDDRG IDVVRNQIKD FASTRQIFSK GFKLIILDEA DAMTNAAQNA LRRVIERYTK NTRFCVLANY AHKLTPALLS R CTRFRFQP LPQEAIERRI ANVLVHEKLK LSPNAEKALI ELSNGDMRRV LNVLQSCKAT LDNPDEDEIS DDVIYECCGA PR PSDLKAV LKSILEDDWG TAHYTLNKVR SAKGLALIDL IEGIVKILED YELQNEETRV HLLTKLADIE YSISKGGNDQ IQG SAVIGA IKASFENETV

UniProtKB: Replication factor C subunit 3

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Macromolecule #4: Replication factor C subunit 2

MacromoleculeName: Replication factor C subunit 2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 39.794473 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MFEGFGPNKK RKISKLAAEQ SLAQQPWVEK YRPKNLDEVT AQDHAVTVLK KTLKSANLPH MLFYGPPGTG KTSTILALTK ELYGPDLMK SRILELNASD ERGISIVREK VKNFARLTVS KPSKHDLENY PCPPYKIIIL DEADSMTADA QSALRRTMET Y SGVTRFCL ...String:
MFEGFGPNKK RKISKLAAEQ SLAQQPWVEK YRPKNLDEVT AQDHAVTVLK KTLKSANLPH MLFYGPPGTG KTSTILALTK ELYGPDLMK SRILELNASD ERGISIVREK VKNFARLTVS KPSKHDLENY PCPPYKIIIL DEADSMTADA QSALRRTMET Y SGVTRFCL ICNYVTRIID PLASRCSKFR FKALDASNAI DRLRFISEQE NVKCDDGVLE RILDISAGDL RRGITLLQSA SK GAQYLGD GKNITSTQVE ELAGVVPHDI LIEIVEKVKS GDFDEIKKYV NTFMKSGWSA ASVVNQLHEY YITNDNFDTN FKN QISWLL FTTDSRLNNG TNEHIQLLNL LVKISQL

UniProtKB: Replication factor C subunit 2

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Macromolecule #5: Replication factor C subunit 5

MacromoleculeName: Replication factor C subunit 5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 39.993582 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSLWVDKYRP KSLNALSHNE ELTNFLKSLS DQPRDLPHLL LYGPNGTGKK TRCMALLESI FGPGVYRLKI DVRQFVTASN RKLELNVVS SPYHLEITPS DMGNNDRIVI QELLKEVAQM EQVDFQDSKD GLAHRYKCVI INEANSLTKD AQAALRRTME K YSKNIRLI ...String:
MSLWVDKYRP KSLNALSHNE ELTNFLKSLS DQPRDLPHLL LYGPNGTGKK TRCMALLESI FGPGVYRLKI DVRQFVTASN RKLELNVVS SPYHLEITPS DMGNNDRIVI QELLKEVAQM EQVDFQDSKD GLAHRYKCVI INEANSLTKD AQAALRRTME K YSKNIRLI MVCDSMSPII APIKSRCLLI RCPAPSDSEI STILSDVVTN ERIQLETKDI LKRIAQASNG NLRVSLLMLE SM ALNNELA LKSSSPIIKP DWIIVIHKLT RKIVKERSVN SLIECRAVLY DLLAHCIPAN IILKELTFSL LDVETLNTTN KSS IIEYSS VFDERLSLGN KAIFHLEGFI AKVMCCLD

UniProtKB: Replication factor C subunit 5

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Macromolecule #6: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 6 / Number of copies: 4 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (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: 1.8 µm / Nominal defocus min: 1.1 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1sxj

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 266496
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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