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- EMDB-25426: Rad24-RFC ADP state -

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Basic information

Entry
Database: EMDB / ID: EMD-25426
TitleRad24-RFC ADP state
Map dataRad24-RFC ADP state
Sample
  • Complex: Rad24-RFC:9-1-1:DNA
    • 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
    • Protein or peptide: Checkpoint protein RAD24
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsDNA damage / DNA replication / DNA sliding clamp / 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 / DNA clamp loader activity / Translesion Synthesis by POLH / Translesion synthesis by REV1 ...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 / DNA clamp loader activity / 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 / mitotic DNA replication checkpoint signaling / reciprocal meiotic recombination / 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 / nucleotide-excision repair / DNA-templated DNA replication / DNA repair / chromatin binding / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
Rad17 P-loop domain / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / 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 / : ...Rad17 P-loop domain / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / 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
Checkpoint protein RAD24 / 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 (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsCastaneda JC / Schrecker M
Funding support3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM107239
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127428
National Institutes of Health/National Cancer Institute (NIH/NCI)CA008748
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Mechanisms of loading and release of the 9-1-1 checkpoint clamp.
Authors: Juan C Castaneda / Marina Schrecker / Dirk Remus / Richard K Hite /
Abstract: Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, ...Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1's recruitment to 5' junctions and reveal new principles of sliding clamp loading.
History
DepositionNov 12, 2021-
Header (metadata) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25426.png

Downloads & links

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Map

FileDownload / File: emd_25426.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRad24-RFC ADP state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 317.184 Å		0.83 Å/pix.
x 384 pix.
= 317.184 Å		0.83 Å/pix.
x 384 pix.
= 317.184 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.8428938 - 2.0203166
Average (Standard dev.)0.00017025422 (±0.03530903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 317.184 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Rad24-RFC:9-1-1:DNA

EntireName: Rad24-RFC:9-1-1:DNA
Components
  • Complex: Rad24-RFC:9-1-1:DNA
    • 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
    • Protein or peptide: Checkpoint protein RAD24
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Rad24-RFC:9-1-1:DNA

SupramoleculeName: Rad24-RFC:9-1-1:DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)

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Macromolecule #1: Replication factor C subunit 4

MacromoleculeName: Replication factor C subunit 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
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 #2: Replication factor C subunit 3

MacromoleculeName: Replication factor C subunit 3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 38.155414 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 KAN

UniProtKB: Replication factor C subunit 3

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Macromolecule #3: Replication factor C subunit 2

MacromoleculeName: Replication factor C subunit 2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
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 #4: Replication factor C subunit 5

MacromoleculeName: Replication factor C subunit 5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
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 #5: Checkpoint protein RAD24

MacromoleculeName: Checkpoint protein RAD24 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 80.096828 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MDSTNLNKRP LLQYSLSSLG SQITKWSSSR PTSPVRKARS TENDFLSKQD TSSILPSIND DGGEQWYEKF KPNCLEQVAI HKRKLKDVQ EALDAMFLPN AKHRILLLSG PSGCSKSTVI KELSKILVPK YRQNSNGTSF RSTPNEHKVT EFRGDCIVND L PQMESFSE ...String:
MDSTNLNKRP LLQYSLSSLG SQITKWSSSR PTSPVRKARS TENDFLSKQD TSSILPSIND DGGEQWYEKF KPNCLEQVAI HKRKLKDVQ EALDAMFLPN AKHRILLLSG PSGCSKSTVI KELSKILVPK YRQNSNGTSF RSTPNEHKVT EFRGDCIVND L PQMESFSE FLKGARYLVM SNLSLILIED LPNVFHIDTR RRFQQLILQW LYSSEPLLPP LVICITECEI PENDNNYRKF GI DYTFSAE TIMNKEILMH PRLKRIKFNP INSTLLKKHL KFICVQNMKM LKEKNKWNKR QEVIDYIAQE TGDIRSAITT LQF WATSSG SLPISTREST ISYFHAIGKV IHGSHSTNND NEMINNLFEN SNNLLSKEDF KLGILENYNT FNKGEFSISD ASSI VDCLS ECDNMNGLPE SNEYGLREVR KTFRNISKQG HNHGTVYFPR EWKVRKLQNS FKVQAEDWLN VSLYKYNAVH SFRNI TLEF GYYAPLIRKC QSYKKKYILY YLKNLPSGSS GPKQTMDKFS DIMKVENGID VVDRIGGPIE ALSVEDGLAP LMDNDS NNC DHLEDQKKER DRRLRMLIDQ YERNVMMAND DLEDEETSFN DDPIVDSDSD NSNNIGNETF GRSDEDESLC EILSQRQ PR KAPVISESLS DSDLEILGLN LEVLFQGPGG DYKDDDDKDY KDDDDKDYKD DDDK

UniProtKB: Checkpoint protein RAD24

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Macromolecule #7: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 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

Concentration1.0 mg/mL
BufferpH: 7.6
Component:
ConcentrationName
25.0 mMHepes-KOH
300.0 mMKOAc
7.0 mMMg(OAc)2
GridModel: Quantifoil R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 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

Particle selectionNumber selected: 4432412
Startup modelType of model: INSILICO MODEL
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 183334
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial model
PDB IDChain

1sxj

source_name: PDB, initial_model_type: experimental model

3a1j

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7ste:
Rad24-RFC ADP state

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