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- PDB-8yjz: Structure of the human endogenous PCNA-FEN1-RNase H2 complex - State D -

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Entry
Database: PDB / ID: 8yjz
TitleStructure of the human endogenous PCNA-FEN1-RNase H2 complex - State D
Components
  • (Ribonuclease H2 subunit ...) x 3
  • Flap endonuclease 1
  • Proliferating cell nuclear antigen
  • downstream DNA
  • parent strand DNA
  • upstream DNA
KeywordsDNA BINDING PROTEIN/DNA / Flap endonuclease 1 / RNase H2 / endogenous DNA / PCNA / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


ribonucleotide metabolic process / ribonuclease H2 complex / flap endonuclease activity / positive regulation of sister chromatid cohesion / telomere maintenance via semi-conservative replication / nucleic acid metabolic process / double-stranded DNA exodeoxyribonuclease activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex ...ribonucleotide metabolic process / ribonuclease H2 complex / flap endonuclease activity / positive regulation of sister chromatid cohesion / telomere maintenance via semi-conservative replication / nucleic acid metabolic process / double-stranded DNA exodeoxyribonuclease activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / 5'-flap endonuclease activity / DNA replication, removal of RNA primer / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / UV protection / Processive synthesis on the lagging strand / regulation of DNA damage checkpoint / PCNA complex / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / HDR through MMEJ (alt-NHEJ) / Removal of the Flap Intermediate from the C-strand / Transcription of E2F targets under negative control by DREAM complex / RNA catabolic process / replisome / 5'-3' exonuclease activity / exonuclease activity / response to L-glutamate / ribonuclease H / histone acetyltransferase binding / DNA polymerase processivity factor activity / G1/S-Specific Transcription / leading strand elongation / replication fork processing / response to dexamethasone / Early Phase of HIV Life Cycle / nuclear replication fork / SUMOylation of DNA replication proteins / POLB-Dependent Long Patch Base Excision Repair / PCNA-Dependent Long Patch Base Excision Repair / RNA nuclease activity / mismatch repair / translesion synthesis / DNA polymerase binding / response to cadmium ion / cyclin-dependent protein kinase holoenzyme complex / estrous cycle / base-excision repair, gap-filling / regulation of G2/M transition of mitotic cell cycle / epithelial cell differentiation / positive regulation of DNA repair / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / male germ cell nucleus / replication fork / nuclear estrogen receptor binding / liver regeneration / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / double-strand break repair via homologous recombination / Translesion Synthesis by POLH / HDR through Homologous Recombination (HRR) / receptor tyrosine kinase binding / Dual Incision in GG-NER / memory / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / positive regulation of fibroblast proliferation / cellular response to UV / RNA-DNA hybrid ribonuclease activity / cellular response to xenobiotic stimulus / double-strand break repair / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / manganese ion binding / heart development / double-stranded DNA binding / fibroblast proliferation / gene expression / endonuclease activity / DNA replication / in utero embryonic development / damaged DNA binding / chromosome, telomeric region / Hydrolases; Acting on ester bonds / nuclear body / negative regulation of gene expression
Similarity search - Function
Ribonuclease H2, subunit C / : / Ribonuclease H2 non-catalytic subunit (Ylr154p-like) / Ribonuclease H2 subunit B, wHTH domain / Ribonuclease H2 subunit B / Rnh202, triple barrel domain / Ydr279p protein family (RNase H2 complex component) wHTH domain / Ydr279p protein triple barrel domain / Ribonuclease HII, helix-loop-helix cap domain superfamily / Ribonuclease H2, subunit A ...Ribonuclease H2, subunit C / : / Ribonuclease H2 non-catalytic subunit (Ylr154p-like) / Ribonuclease H2 subunit B, wHTH domain / Ribonuclease H2 subunit B / Rnh202, triple barrel domain / Ydr279p protein family (RNase H2 complex component) wHTH domain / Ydr279p protein triple barrel domain / Ribonuclease HII, helix-loop-helix cap domain superfamily / Ribonuclease H2, subunit A / Ribonuclease HII/HIII / Ribonuclease HII/HIII domain / Ribonuclease HII / Ribonuclease (RNase) H type-2 domain profile. / Flap endonuclease 1 / XPG protein signature 2. / XPG conserved site / XPG protein signature 1. / XPG/Rad2 endonuclease / XPG, N-terminal / XPG-I domain / XPG N-terminal domain / XPG I-region / Xeroderma pigmentosum G I-region / Xeroderma pigmentosum G N-region / Proliferating cell nuclear antigen signature 2. / Helix-hairpin-helix motif, class 2 / Helix-hairpin-helix class 2 (Pol1 family) motifs / 5'-3' exonuclease, C-terminal domain superfamily / 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 / PIN-like domain superfamily / : / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Ribonuclease H2 subunit A / Proliferating cell nuclear antigen / Flap endonuclease 1 / Ribonuclease H2 subunit B / Ribonuclease H2 subunit C
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.15 Å
AuthorsTian, Y. / Gao, N.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: EMBO J / Year: 2024
Title: Structural insight into Okazaki fragment maturation mediated by PCNA-bound FEN1 and RNaseH2.
Authors: Yuhui Tian / Ningning Li / Qing Li / Ning Gao /
Abstract: PCNA is a master coordinator of many DNA-metabolic events. During DNA replication, the maturation of Okazaki fragments involves at least four DNA enzymes, all of which contain PCNA-interacting motifs. ...PCNA is a master coordinator of many DNA-metabolic events. During DNA replication, the maturation of Okazaki fragments involves at least four DNA enzymes, all of which contain PCNA-interacting motifs. However, the temporal relationships and functional modulations between these PCNA-binding proteins are unclear. Here, we developed a strategy to purify endogenous PCNA-containing complexes from native chromatin, and characterized their structures using cryo-EM. Two structurally resolved classes (PCNA-FEN1 and PCNA-FEN1-RNaseH2 complexes) have captured a series of 3D snapshots for the primer-removal steps of Okazaki fragment maturation. These structures show that product release from FEN1 is a rate-liming step. Furthermore, both FEN1 and RNaseH2 undergo continuous conformational changes on PCNA that result in constant fluctuations in the bending angle of substrate DNA at the nick site, implying that these enzymes could regulate each other through conformational modulation of the bound DNA. The structures of the PCNA-FEN1-RNaseH2 complex confirm the toolbelt function of PCNA and suggests a potential unrecognized role of RNaseH2, as a dsDNA binding protein, in promoting the 5'-flap cleaving activity of FEN1.
History
DepositionMar 3, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Proliferating cell nuclear antigen
D: Flap endonuclease 1
H: Ribonuclease H2 subunit A
G: Ribonuclease H2 subunit B
I: Ribonuclease H2 subunit C
C: Proliferating cell nuclear antigen
A: Proliferating cell nuclear antigen
J: upstream DNA
E: parent strand DNA
F: downstream DNA


Theoretical massNumber of molelcules
Total (without water)235,45510
Polymers235,45510
Non-polymers00
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 BCAD

#1: Protein Proliferating cell nuclear antigen / PCNA / Cyclin


Mass: 28795.752 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PCNA / Production host: Homo sapiens (human) / References: UniProt: P12004
#2: Protein Flap endonuclease 1 / FEN-1 / DNase IV / Flap structure-specific endonuclease 1 / Maturation factor 1 / MF1 / hFEN-1


Mass: 42661.051 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
References: UniProt: P39748, Hydrolases; Acting on ester bonds

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Ribonuclease H2 subunit ... , 3 types, 3 molecules HGI

#3: Protein Ribonuclease H2 subunit A / RNase H2 subunit A / Aicardi-Goutieres syndrome 4 protein / AGS4 / RNase H(35) / Ribonuclease HI ...RNase H2 subunit A / Aicardi-Goutieres syndrome 4 protein / AGS4 / RNase H(35) / Ribonuclease HI large subunit / RNase HI large subunit / Ribonuclease HI subunit A


Mass: 33431.844 Da / Num. of mol.: 1 / Fragment: subunit A / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O75792, ribonuclease H
#4: Protein Ribonuclease H2 subunit B / RNase H2 subunit B / Aicardi-Goutieres syndrome 2 protein / AGS2 / Deleted in lymphocytic leukemia ...RNase H2 subunit B / Aicardi-Goutieres syndrome 2 protein / AGS2 / Deleted in lymphocytic leukemia 8 / Ribonuclease HI subunit B


Mass: 35195.727 Da / Num. of mol.: 1 / Fragment: subunit B / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q5TBB1
#5: Protein Ribonuclease H2 subunit C / RNase H2 subunit C / Aicardi-Goutieres syndrome 3 protein / AGS3 / RNase H1 small subunit / ...RNase H2 subunit C / Aicardi-Goutieres syndrome 3 protein / AGS3 / RNase H1 small subunit / Ribonuclease HI subunit C


Mass: 17862.137 Da / Num. of mol.: 1 / Fragment: subunit C / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q8TDP1

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DNA chain , 3 types, 3 molecules JEF

#6: DNA chain upstream DNA


Mass: 6101.997 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
#7: DNA chain parent strand DNA


Mass: 9565.299 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
#8: DNA chain downstream DNA


Mass: 4249.797 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)

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Details

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

ComponentName: endogenous state D PCNA-DNA-FEN1-RNase H2 complex / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Homo sapiens (human)
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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 2000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 5.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13906 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00415342
ELECTRON MICROSCOPYf_angle_d1.02721007
ELECTRON MICROSCOPYf_dihedral_angle_d18.8652500
ELECTRON MICROSCOPYf_chiral_restr0.062394
ELECTRON MICROSCOPYf_plane_restr0.0072480

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