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- EMDB-40522: Origin Recognition Complex Associated (ORCA) protein bound to H4K... -

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Basic information

Entry
Database: EMDB / ID: EMD-40522
TitleOrigin Recognition Complex Associated (ORCA) protein bound to H4K20me3-nucleosome
Map dataunsharpened map
Sample
  • Complex: ORCA-nucleosome (H4K20me3) complex
    • Complex: Leucine-rich repeat and WD repeat-containing protein 1, Origin recognition complex subunit 2
      • Protein or peptide: Leucine-rich repeat and WD repeat-containing protein 1
      • Protein or peptide: Origin recognition complex subunit 2
    • Complex: Histone H3.2, Histone H4, Histone H2A, Histone H2B, Widom 601 DNA
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A
      • Protein or peptide: Histone H2B
      • DNA: Widom 601 DNA
      • DNA: Widom 601 DNA
Keywordschromatin binding / ORC binding / nucleosome / REPLICATION
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 / establishment of protein localization to chromatin / inner kinetochore / 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 / establishment of protein localization to chromatin / inner kinetochore / 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. / Leucine-rich repeat / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site ...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. / Leucine-rich repeat / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBleichert F / Ekundayo BE
Funding support United States, European Union, 2 items
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, 2023-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40522.png

Downloads & links

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Map

FileDownload / File: emd_40522.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened map
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.4347084 - 1.1434567
Average (Standard dev.)0.00097216415 (±0.044772185)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: DeepEMhancer map

Fileemd_40522_additional_1.map
AnnotationDeepEMhancer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: sharpened map

Fileemd_40522_additional_2.map
Annotationsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap

Fileemd_40522_half_map_1.map
Annotationhalfmap
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap

Fileemd_40522_half_map_2.map
Annotationhalfmap
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ORCA-nucleosome (H4K20me3) complex

EntireName: ORCA-nucleosome (H4K20me3) complex
Components
  • Complex: ORCA-nucleosome (H4K20me3) complex
    • Complex: Leucine-rich repeat and WD repeat-containing protein 1, Origin recognition complex subunit 2
      • Protein or peptide: Leucine-rich repeat and WD repeat-containing protein 1
      • Protein or peptide: Origin recognition complex subunit 2
    • Complex: Histone H3.2, Histone H4, Histone H2A, Histone H2B, Widom 601 DNA
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A
      • Protein or peptide: Histone H2B
      • DNA: Widom 601 DNA
      • DNA: Widom 601 DNA

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Supramolecule #1: ORCA-nucleosome (H4K20me3) complex

SupramoleculeName: ORCA-nucleosome (H4K20me3) complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8

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Supramolecule #2: Leucine-rich repeat and WD repeat-containing protein 1, Origin re...

SupramoleculeName: Leucine-rich repeat and WD repeat-containing protein 1, Origin recognition complex subunit 2
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #8
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Supramolecule #3: Histone H3.2, Histone H4, Histone H2A, Histone H2B, Widom 601 DNA

SupramoleculeName: Histone H3.2, Histone H4, Histone H2A, Histone H2B, Widom 601 DNA
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#7
Source (natural)Organism: Xenopus laevis (African clawed frog)

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Macromolecule #1: Leucine-rich repeat and WD repeat-containing protein 1

MacromoleculeName: Leucine-rich repeat and WD repeat-containing protein 1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 71.651547 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: SNAMAPLTPQ LLLQRGRPKT DRLGKIQSLN LSGLELLSEH LDPNLLGRLK KLKELDLSNN LLETLPANLG LSHLRILRCT NNQLGDVTT LHQFPELEEL SLEGNPFLTV SDNLKVSFLL PKLRKVNGKD TASTCSQVES LDRELTNRVT ALWQKFIATV N PEEEANKV ...String:
SNAMAPLTPQ LLLQRGRPKT DRLGKIQSLN LSGLELLSEH LDPNLLGRLK KLKELDLSNN LLETLPANLG LSHLRILRCT NNQLGDVTT LHQFPELEEL SLEGNPFLTV SDNLKVSFLL PKLRKVNGKD TASTCSQVES LDRELTNRVT ALWQKFIATV N PEEEANKV QADFMRSAVR DVRYGPESLF EFTQWRVRMI AEELVASGGA QAHEANASVE HPQAAVGSKL RARKVTKRPD DD EISLPPT KRVRASPPAQ TEDSPMDAAG GQAALHLEPL HFLQCHSRNN SPKDLETQLW ACAFEPAREE GHSGATSQTV ATC GGEAVC VIDCQTGLVL HKYKVPGEEF FSVAWTALTV ATQAGHKKRW NMLAAAGLRG MVRLLHVRAG FCCSVIRAHK KAIA TLCFS PTHETHLFTA SYDKRIILWD IGVPNHDYKF QASQLLTLNC SSVPLRLCPV ATCPDSFLLA GCEGGCGCWD VRLDQ PQKQ RVCEVNFVFS GDSEVSGQRV DGLAFVNEDV VASKGSGQGT IYLWSWSQTW ASRGSQSVLP VVILAQLQWS PTSLAY FSL STCPDKNLVL CGDEEGSVWI YDVEHLLKQP PPLETTLQPP TQILKWPQPV ALGQPVTKTM VNTVVANAAF TYLTALT DS NIVSIWKTC

UniProtKB: Leucine-rich repeat and WD repeat-containing protein 1

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Macromolecule #2: Histone H3.2

MacromoleculeName: Histone H3.2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 15.30393 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAV MALQEASEAY LVALFEDTNL CAIHAKRVTI MPKDIQLARR IRGERA

UniProtKB: Histone H3.2

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Macromolecule #3: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 11.32335 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKGGKGL GKGGAKRHR(ML3) VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHA KRK TVTAMDVVYA LKRQGRTLYG FGG

UniProtKB: Histone H4

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Macromolecule #4: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 13.962241 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKQGGKT RAKAKTRSSR AGLQFPVGRV HRLLRKGNYA ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAVRN DEELNKLLGR VTIAQGGVLP NIQSVLLPKK TESAKSAKSK

UniProtKB: Histone H2A type 1

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Macromolecule #5: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 13.524752 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AKSAPAPKKG SKKAVTKTQK KDGKKRRKTR KESYAIYVYK VLKQVHPDTG ISSKAMSIMN SFVNDVFERI AGEASRLAHY NKRSTITSR EIQTAVRLLL PGELAKHAVS EGTKAVTKYT SAK

UniProtKB: Histone H2B 1.1

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Macromolecule #8: Origin recognition complex subunit 2

MacromoleculeName: Origin recognition complex subunit 2 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 11.570101 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
SNAMSTLRLK EAKVPSVQFV GDDDVLSHIL DREGGTKLKK EKVQLLVNPQ KVIKKAECEL EKSDLEVLED QNYVEVLGRN IQESLGNGS AVDGRNKVYS FQHR

UniProtKB: Origin recognition complex subunit 2

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Macromolecule #6: Widom 601 DNA

MacromoleculeName: Widom 601 DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 46.998945 KDa
SequenceString: (DA)(DT)(DC)(DC)(DT)(DG)(DG)(DA)(DG)(DA) (DA)(DT)(DC)(DC)(DC)(DG)(DG)(DT)(DG)(DC) (DC)(DG)(DA)(DG)(DG)(DC)(DC)(DG)(DC) (DT)(DC)(DA)(DA)(DT)(DT)(DG)(DG)(DT)(DC) (DG) (DT)(DA)(DG)(DA)(DC)(DA) ...String:
(DA)(DT)(DC)(DC)(DT)(DG)(DG)(DA)(DG)(DA) (DA)(DT)(DC)(DC)(DC)(DG)(DG)(DT)(DG)(DC) (DC)(DG)(DA)(DG)(DG)(DC)(DC)(DG)(DC) (DT)(DC)(DA)(DA)(DT)(DT)(DG)(DG)(DT)(DC) (DG) (DT)(DA)(DG)(DA)(DC)(DA)(DG)(DC) (DT)(DC)(DT)(DA)(DG)(DC)(DA)(DC)(DC)(DG) (DC)(DT) (DT)(DA)(DA)(DA)(DC)(DG)(DC) (DA)(DC)(DG)(DT)(DA)(DC)(DG)(DC)(DG)(DC) (DT)(DG)(DT) (DC)(DC)(DC)(DC)(DC)(DG) (DC)(DG)(DT)(DT)(DT)(DT)(DA)(DA)(DC)(DC) (DG)(DC)(DC)(DA) (DA)(DG)(DG)(DG)(DG) (DA)(DT)(DT)(DA)(DC)(DT)(DC)(DC)(DC)(DT) (DA)(DG)(DT)(DC)(DT) (DC)(DC)(DA)(DG) (DG)(DC)(DA)(DC)(DG)(DT)(DG)(DT)(DC)(DA) (DG)(DA)(DT)(DA)(DT)(DA) (DT)(DA)(DC) (DA)(DT)(DC)(DC)(DT)(DG)(DT)(DG)(DA)(DT)

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Macromolecule #7: Widom 601 DNA

MacromoleculeName: Widom 601 DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 47.457234 KDa
SequenceString: (DA)(DT)(DC)(DA)(DC)(DA)(DG)(DG)(DA)(DT) (DG)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT) (DG)(DA)(DC)(DA)(DC)(DG)(DT)(DG)(DC) (DC)(DT)(DG)(DG)(DA)(DG)(DA)(DC)(DT)(DA) (DG) (DG)(DG)(DA)(DG)(DT)(DA) ...String:
(DA)(DT)(DC)(DA)(DC)(DA)(DG)(DG)(DA)(DT) (DG)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT) (DG)(DA)(DC)(DA)(DC)(DG)(DT)(DG)(DC) (DC)(DT)(DG)(DG)(DA)(DG)(DA)(DC)(DT)(DA) (DG) (DG)(DG)(DA)(DG)(DT)(DA)(DA)(DT) (DC)(DC)(DC)(DC)(DT)(DT)(DG)(DG)(DC)(DG) (DG)(DT) (DT)(DA)(DA)(DA)(DA)(DC)(DG) (DC)(DG)(DG)(DG)(DG)(DG)(DA)(DC)(DA)(DG) (DC)(DG)(DC) (DG)(DT)(DA)(DC)(DG)(DT) (DG)(DC)(DG)(DT)(DT)(DT)(DA)(DA)(DG)(DC) (DG)(DG)(DT)(DG) (DC)(DT)(DA)(DG)(DA) (DG)(DC)(DT)(DG)(DT)(DC)(DT)(DA)(DC)(DG) (DA)(DC)(DC)(DA)(DA) (DT)(DT)(DG)(DA) (DG)(DC)(DG)(DG)(DC)(DC)(DT)(DC)(DG)(DG) (DC)(DA)(DC)(DC)(DG)(DG) (DG)(DA)(DT) (DT)(DC)(DT)(DC)(DC)(DA)(DG)(DG)(DA)(DT)

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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
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 51.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 31420
FSC plot (resolution estimation)

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