[English] 日本語
Yorodumi
- PDB-8fs6: Structure of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8fs6
TitleStructure of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 4 (partially closed 9-1-1 and stably bound chamber DNA)
Components
  • (DNA damage checkpoint control protein ...) x 2
  • (Primer strand ...) x 2
  • (Replication factor C subunit ...) x 4
  • Checkpoint protein RAD24
  • DDC1 isoform 1
  • Template strand
KeywordsCELL CYCLE/DNA / DNA damage repair / Rad24-RFC / 9-1-1 clamp / DNA clamp / alternative clamp loader / DNA damage signaling / DNA BINDING PROTEIN-DNA complex / CELL CYCLE-DNA complex
Function / homology
Function and homology information


meiotic DNA integrity checkpoint signaling / checkpoint clamp complex / DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Ctf18 RFC-like complex / Rad17 RFC-like complex / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / DNA clamp loader activity ...meiotic DNA integrity checkpoint signaling / checkpoint clamp complex / DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Ctf18 RFC-like complex / Rad17 RFC-like complex / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / DNA clamp loader activity / telomere maintenance via recombination / 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 / recombinational repair / mitotic intra-S DNA damage checkpoint signaling / mitotic sister chromatid cohesion / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / subtelomeric heterochromatin formation / mismatch repair / 3'-5' exonuclease activity / telomere maintenance / meiotic cell cycle / DNA damage checkpoint signaling / cellular response to ionizing radiation / nucleotide-excision repair / double-strand break repair via homologous recombination / DNA-templated DNA replication / double-strand break repair / site of double-strand break / double-stranded DNA binding / chromosome, telomeric region / damaged DNA binding / DNA repair / chromatin binding / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
Ddc1 / DNA damage checkpoint control protein Rad17 / Rad17 P-loop domain / Checkpoint protein Hus1/Mec3 / Hus1-like protein / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / Rad1/Rec1/Rad17 / Rad9/Ddc1 / Repair protein Rad1/Rec1/Rad17 ...Ddc1 / DNA damage checkpoint control protein Rad17 / Rad17 P-loop domain / Checkpoint protein Hus1/Mec3 / Hus1-like protein / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / Rad1/Rec1/Rad17 / Rad9/Ddc1 / Repair protein Rad1/Rec1/Rad17 / 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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DDC1 isoform 1 / DNA damage checkpoint control protein RAD17 / Checkpoint protein RAD24 / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 4 ...ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DDC1 isoform 1 / DNA damage checkpoint control protein RAD17 / Checkpoint protein RAD24 / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 4 / Replication factor C subunit 2 / DNA damage checkpoint control protein MEC3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsZheng, F. / Georgescu, R. / Yao, Y.N. / 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: bioRxiv / Year: 2023
Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification to biology.
Authors: Fengwei Zheng / Roxana E Georgescu / Nina Y Yao / Michael E O'Donnell / Huilin Li /
Abstract: Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the ...Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the well-established internal chamber and into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' DNA end, thus presumably leaving 9-1-1 on a 3' ss/ds DNA after Rad24-RFC ejects from the 5' gap end and may explain reports of 9-1-1 directly functioning in DNA repair with various TLS polymerases, in addition to signaling the ATR kinase. To gain a deeper understanding of 9-1-1 loading at gaps we report high-resolution structures of Rad24-RFC during loading of 9-1-1 onto 10-nt and 5-nt gapped DNAs. At a 10-nt gap we captured five Rad24-RFC-9-1-1 loading intermediates in which the 9-1-1 DNA entry gate varies from fully open to fully closed around DNA using ATPγS, supporting the emerging view that ATP hydrolysis is not needed for clamp opening/closing, but instead for dissociation of the loader from the clamp encircling DNA. The structure of Rad24-RFC-9-1-1 at a 5-nt gap shows a 180° axially rotated 3'-dsDNA which orients the template strand to bridge the 3'- and 5'- junctions with a minimum 5-nt ssDNA. The structures reveal a unique loop on Rad24 that limits the length of dsDNA in the inner chamber, and inability to melt DNA ends unlike RFC, thereby explaining Rad24-RFC's preference for a preexisting ssDNA gap and suggesting a direct role in gap repair in addition to its checkpoint role.
History
DepositionJan 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Checkpoint protein RAD24
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: DNA damage checkpoint control protein MEC3
G: DNA damage checkpoint control protein RAD17
H: DDC1 isoform 1
I: Template strand
J: Primer strand 1
K: Primer strand 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)415,81220
Polymers413,19411
Non-polymers2,6179
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, 2D averages showed the protein-DNA complex formed successfully
TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

-
Protein , 2 types, 2 molecules AH

#1: Protein Checkpoint protein RAD24


Mass: 62792.496 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAD24, YER173W, SYGP-ORF60 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32641
#8: Protein DDC1 isoform 1


Mass: 69787.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DDC1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4BUG7

-
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

-
DNA damage checkpoint control protein ... , 2 types, 2 molecules FG

#6: Protein DNA damage checkpoint control protein MEC3


Mass: 53207.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MEC3, PIP3, PSO9, YLR288C, L8003.15 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02574
#7: Protein DNA damage checkpoint control protein RAD17


Mass: 45637.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAD17, GI527_G0005737 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4BW58

-
DNA chain , 1 types, 1 molecules I

#9: DNA chain Template strand


Mass: 15389.855 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

-
Primer strand ... , 2 types, 2 molecules JK

#10: DNA chain Primer strand 1


Mass: 6464.172 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#11: DNA chain Primer strand 2


Mass: 6084.940 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

-
Non-polymers , 3 types, 9 molecules

#12: 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
#13: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#14: 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

-
Details

Has ligand of interestN

-
Experimental details

-
Experiment

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

-
Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Rad24-RFC-911 clamp-DNACOMPLEX#6-#110MULTIPLE SOURCES
2DNACOMPLEX#9-#111MULTIPLE SOURCESsynthetic DNA
3Proteins complexCOMPLEX#6-#81RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12synthetic construct (others)32630
23Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

-
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: 1900 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

-
Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 191448 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00522765
ELECTRON MICROSCOPYf_angle_d0.7431009
ELECTRON MICROSCOPYf_dihedral_angle_d14.9013417
ELECTRON MICROSCOPYf_chiral_restr0.0423584
ELECTRON MICROSCOPYf_plane_restr0.0053708

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbjlvh1.pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more