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- PDB-8siy: Origin Recognition Complex Associated (ORCA) protein bound to H4K... -

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Basic information

Entry
Database: PDB / ID: 8siy
TitleOrigin Recognition Complex Associated (ORCA) protein bound to H4K20me3-nucleosome
Components
  • (Widom 601 DNA) x 2
  • Histone H2A
  • Histone H2B
  • Histone H3.2
  • Histone H4
  • Leucine-rich repeat and WD repeat-containing protein 1
  • Origin recognition complex subunit 2
KeywordsREPLICATION / chromatin binding / ORC binding / nucleosome
Function / homology
Function and homology information


Activation of ATR in response to replication stress / Assembly of the ORC complex at the origin of replication / CDC6 association with the ORC:origin complex / Activation of the pre-replicative complex / Orc1 removal from chromatin / origin recognition complex / inner kinetochore / establishment of protein localization to chromatin / nuclear origin of replication recognition complex / methyl-CpG binding ...Activation of ATR in response to replication stress / Assembly of the ORC complex at the origin of replication / CDC6 association with the ORC:origin complex / Activation of the pre-replicative complex / Orc1 removal from chromatin / origin recognition complex / inner kinetochore / establishment of protein localization to chromatin / nuclear origin of replication recognition complex / methyl-CpG binding / DNA replication origin binding / DNA replication initiation / heterochromatin / pericentric heterochromatin / methylated histone binding / kinetochore / structural constituent of chromatin / nucleosome / chromatin organization / DNA replication / chromosome, telomeric region / cytoskeleton / protein heterodimerization activity / centrosome / chromatin binding / chromatin / nucleolus / DNA binding / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
Origin recognition complex subunit 2 / Origin recognition complex, subunit 2 / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. ...Origin recognition complex subunit 2 / Origin recognition complex, subunit 2 / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Leucine-rich repeat / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / Leucine-rich repeat domain superfamily / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Leucine-rich repeat and WD repeat-containing protein 1 / Histone H4 / Histone H2B 1.1 / Histone H2A type 1 / Histone H3.2 / Origin recognition complex subunit 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Xenopus laevis (African clawed frog)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBleichert, F. / Ekundayo, B.E.
Funding support United States, European Union, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM141313 United States
European Research Council (ERC)ERC-STG-757909European Union
CitationJournal: EMBO J / Year: 2023
Title: A dual role for the chromatin reader ORCA/LRWD1 in targeting the origin recognition complex to chromatin.
Authors: Sumon Sahu / Babatunde E Ekundayo / Ashish Kumar / Franziska Bleichert /
Abstract: Eukaryotic cells use chromatin marks to regulate the initiation of DNA replication. The origin recognition complex (ORC)-associated protein ORCA plays a critical role in heterochromatin replication ...Eukaryotic cells use chromatin marks to regulate the initiation of DNA replication. The origin recognition complex (ORC)-associated protein ORCA plays a critical role in heterochromatin replication in mammalian cells by recruiting the initiator ORC, but the underlying mechanisms remain unclear. Here, we report crystal and cryo-electron microscopy structures of ORCA in complex with ORC's Orc2 subunit and nucleosomes, establishing that ORCA orchestrates ternary complex assembly by simultaneously recognizing a highly conserved peptide sequence in Orc2, nucleosomal DNA, and repressive histone trimethylation marks through an aromatic cage. Unexpectedly, binding of ORCA to nucleosomes prevents chromatin array compaction in a manner that relies on H4K20 trimethylation, a histone modification critical for heterochromatin replication. We further show that ORCA is necessary and sufficient to specifically recruit ORC into chromatin condensates marked by H4K20 trimethylation, providing a paradigm for studying replication initiation in specific chromatin contexts. Collectively, our findings support a model in which ORCA not only serves as a platform for ORC recruitment to nucleosomes bearing specific histone marks but also helps establish a local chromatin environment conducive to subsequent MCM2-7 loading.
History
DepositionApr 17, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 2, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Sep 27, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Leucine-rich repeat and WD repeat-containing protein 1
C: Histone H3.2
D: Histone H4
E: Histone H2A
F: Histone H2B
G: Histone H3.2
H: Histone H4
I: Histone H2A
J: Histone H2B
K: Widom 601 DNA
L: Widom 601 DNA
B: Origin recognition complex subunit 2


Theoretical massNumber of molelcules
Total (without water)285,90612
Polymers285,90612
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 6 types, 10 molecules ACGDHEIFJB

#1: Protein Leucine-rich repeat and WD repeat-containing protein 1 / Origin recognition complex-associated protein


Mass: 71651.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Lrwd1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A140UHX1
#2: Protein Histone H3.2 / Histone H3


Mass: 15303.930 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P84233
#3: Protein Histone H4 /


Mass: 11323.350 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: LOC121398084 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J1LTD2
#4: Protein Histone H2A / / Histone H2A type 1


Mass: 13962.241 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P06897
#5: Protein Histone H2B / / H2B1.1


Mass: 13524.752 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P02281
#8: Protein Origin recognition complex subunit 2 /


Mass: 11570.101 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Orc2, Orc2l / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q75PQ8

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DNA chain , 2 types, 2 molecules KL

#6: DNA chain Widom 601 DNA


Mass: 46998.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#7: DNA chain Widom 601 DNA


Mass: 47457.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Details

Has ligand of interestY

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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
1ORCA-nucleosome (H4K20me3) complexCOMPLEXall0RECOMBINANT
2Leucine-rich repeat and WD repeat-containing protein 1, Origin recognition complex subunit 2COMPLEX#1, #81RECOMBINANT
3Histone H3.2, Histone H4, Histone H2A, Histone H2B, Widom 601 DNACOMPLEX#2-#71RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Xenopus laevis (African clawed frog)8355
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31420 / Symmetry type: POINT
RefinementCross valid method: NONE

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