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- PDB-8siu: Origin Recognition Complex Associated (ORCA) protein bound to Orc2 -

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Basic information

Entry
Database: PDB / ID: 8siu
TitleOrigin Recognition Complex Associated (ORCA) protein bound to Orc2
Components
  • Leucine-rich repeat and WD repeat-containing protein 1
  • Origin recognition complex subunit 2
KeywordsPROTEIN BINDING / chromatin binding / ORC binding
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 / chromatin organization / DNA replication / chromosome, telomeric region / cytoskeleton / centrosome / chromatin binding / chromatin / nucleolus / 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 / Leucine-rich repeat domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat ...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 / Leucine-rich repeat domain superfamily / 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 / Origin recognition complex subunit 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.8 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
B: Origin recognition complex subunit 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,9934
Polymers53,8012
Non-polymers1922
Water5,783321
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2130 Å2
ΔGint-25 kcal/mol
Surface area16540 Å2
MethodPISA
Unit cell
Length a, b, c (Å)91.023, 135.431, 78.567
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Components on special symmetry positions
IDModelComponents
11A-702-

SO4

21A-982-

HOH

31A-1083-

HOH

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Components

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


Mass: 42230.711 Da / Num. of mol.: 1 / Mutation: K646R,T647R
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 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
#3: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 321 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.25 Å3/Da / Density % sol: 45.34 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / Details: 1.2 M ammonium sulfate, 100 mM MES pH 5.3

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 0.979092 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Oct 2, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979092 Å / Relative weight: 1
ReflectionResolution: 1.8→45.51 Å / Num. obs: 45458 / % possible obs: 99.8 % / Redundancy: 39 % / Biso Wilson estimate: 22.28 Å2 / CC1/2: 0.999 / Net I/σ(I): 14.8
Reflection shellResolution: 1.8→1.83 Å / Mean I/σ(I) obs: 1.1 / Num. unique obs: 2577 / CC1/2: 0.505 / % possible all: 97.4

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: SAD / Resolution: 1.8→45.51 Å / SU ML: 0.1869 / Cross valid method: FREE R-VALUE / σ(F): 0.01 / Phase error: 21.1448
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2032 1847 4.4 %
Rwork0.1697 40157 -
obs0.1712 42004 92.3 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 31.85 Å2
Refinement stepCycle: LAST / Resolution: 1.8→45.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2985 0 10 321 3316
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00983119
X-RAY DIFFRACTIONf_angle_d1.09084265
X-RAY DIFFRACTIONf_chiral_restr0.0625486
X-RAY DIFFRACTIONf_plane_restr0.008545
X-RAY DIFFRACTIONf_dihedral_angle_d13.44151100
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.850.38981130.31222410X-RAY DIFFRACTION73.62
1.85-1.90.27181220.25732638X-RAY DIFFRACTION79.47
1.9-1.960.28491300.22662819X-RAY DIFFRACTION85.4
1.96-2.030.26131350.22063061X-RAY DIFFRACTION91.84
2.03-2.110.25281430.19563127X-RAY DIFFRACTION94.76
2.11-2.210.19931460.16793137X-RAY DIFFRACTION94.1
2.21-2.330.20951480.15923134X-RAY DIFFRACTION94.47
2.33-2.470.22161440.15813168X-RAY DIFFRACTION94.85
2.47-2.660.20661480.16273192X-RAY DIFFRACTION96
2.66-2.930.19231510.16663272X-RAY DIFFRACTION97.27
2.93-3.350.21391490.15893306X-RAY DIFFRACTION98.21
3.35-4.220.17391550.14193385X-RAY DIFFRACTION99.55
4.22-45.510.16511630.16343508X-RAY DIFFRACTION99.35

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