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- PDB-8zp5: Cryo-EM structure of origin recognition complex (Orc5 basic patch... -

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Basic information

Entry
Database: PDB / ID: 8zp5
TitleCryo-EM structure of origin recognition complex (Orc5 basic patch mutations) with ARS1 DNA bound
Components
  • (Origin recognition complex subunit ...) x 6
  • DNA (34-MER)
  • DNA (35-MER)
KeywordsREPLICATION
Function / homology
Function and homology information


CDC6 association with the ORC:origin complex / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / Assembly of the ORC complex at the origin of replication / nuclear origin of replication recognition complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / nucleosome organization ...CDC6 association with the ORC:origin complex / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / Assembly of the ORC complex at the origin of replication / nuclear origin of replication recognition complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / nucleosome organization / DNA replication preinitiation complex / mitotic DNA replication checkpoint signaling / silent mating-type cassette heterochromatin formation / Orc1 removal from chromatin / regulation of DNA replication / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / nucleosome binding / chromosome, telomeric region / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / metal ion binding / nucleus
Similarity search - Function
Origin recognition complex, subunit 6, fungi / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Origin recognition complex subunit 4 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex subunit 4, C-terminal / Origin recognition complex subunit 3, winged helix C-terminal ...Origin recognition complex, subunit 6, fungi / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Origin recognition complex subunit 4 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex subunit 4, C-terminal / Origin recognition complex subunit 3, winged helix C-terminal / Origin recognition complex subunit 3, N-terminal / : / : / Origin recognition complex (ORC) subunit 3 N-terminus / Origin recognition complex (ORC) subunit 4 C-terminus / Origin recognition complex (ORC) subunit 5 C-terminus / Origin recognition complex winged helix C-terminal / ORC5, lid domain / Orc1-like, AAA ATPase domain / : / : / Origin recognition complex subunit 2 RecA-like domain / ORC2 WHD / AAA ATPase domain / Origin recognition complex, subunit 2 / AAA lid domain / AAA lid domain / : / Bromo adjacent homology domain / BAH domain / Bromo adjacent homology (BAH) domain / Bromo adjacent homology (BAH) domain superfamily / BAH domain profile. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / EF-Hand 1, calcium-binding site / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Origin recognition complex subunit 2 / Origin recognition complex subunit 6 / Origin recognition complex subunit 5 / Origin recognition complex subunit 1 / Origin recognition complex subunit 3 / Origin recognition complex subunit 4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsLam, W.H. / Yu, D. / Dang, S. / Zhai, Y.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: DNA bending mediated by ORC is essential for replication licensing in budding yeast.
Authors: Wai Hei Lam / Daqi Yu / Qiongdan Zhang / Yuhan Lin / Ningning Li / Jian Li / Yue Wu / Yingyi Zhang / Ning Gao / Bik Kwoon Tye / Yuanliang Zhai / Shangyu Dang /
Abstract: In eukaryotes, the origin recognition complex (ORC) promotes the assembly of minichromosome maintenance 2 to 7 complexes into a head-to-head double hexamer at origin DNA in a process known as ...In eukaryotes, the origin recognition complex (ORC) promotes the assembly of minichromosome maintenance 2 to 7 complexes into a head-to-head double hexamer at origin DNA in a process known as replication licensing. In this study, we present a series of cryoelectron microscopy structures of yeast ORC mutants in complex with origin DNA. We show that Orc6, the smallest subunit of ORC, utilizes its transcription factor II B-B domain to orchestrate the sequential binding of ORC to origin DNA. In addition, Orc6 plays the role of a scaffold by stabilizing the basic patch (BP) of Orc5 for ORC to capture and bend origin DNA. Importantly, disrupting DNA bending through mutating three key residues in Orc5-BP impairs ORC's ability to promote replication initiation at two points during the pre-RC assembly process. This study dissects the multifaceted role of Orc6 in orchestrating ORC's activities on DNA and underscores the vital role of DNA bending by ORC in replication licensing.
History
DepositionMay 29, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 16, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Origin recognition complex subunit 2
E: Origin recognition complex subunit 5
F: Origin recognition complex subunit 6
G: DNA (35-MER)
H: DNA (34-MER)
A: Origin recognition complex subunit 1
C: Origin recognition complex subunit 3
D: Origin recognition complex subunit 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)463,42614
Polymers461,7838
Non-polymers1,6436
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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Origin recognition complex subunit ... , 6 types, 6 molecules BEFACD

#1: Protein Origin recognition complex subunit 2 / Origin recognition complex 71 kDa subunit


Mass: 71342.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: ORC2 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32833
#2: Protein Origin recognition complex subunit 5 / Origin recognition complex 53 kDa subunit


Mass: 55116.832 Da / Num. of mol.: 1 / Mutation: R360A, R366A, K367A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: ORC5 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P50874
#3: Protein Origin recognition complex subunit 6 / ACS-associated protein 1 / Origin recognition complex 50 kDa subunit


Mass: 50369.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: ORC6 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38826
#6: Protein Origin recognition complex subunit 1 / Origin recognition complex 120 kDa subunit


Mass: 104546.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: ORC1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54784
#7: Protein Origin recognition complex subunit 3 / Origin recognition complex 62 kDa subunit


Mass: 72161.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: ORC3 / Variant: S / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54790
#8: Protein Origin recognition complex subunit 4 / Origin recognition complex 56 kDa subunit


Mass: 60772.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: ORC4 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54791

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DNA chain , 2 types, 2 molecules GH

#4: DNA chain DNA (35-MER)


Mass: 23708.256 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#5: DNA chain DNA (34-MER)


Mass: 23766.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Non-polymers , 2 types, 6 molecules

#9: 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.: 3 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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: Origin recognition complex with Orc5 basic patch mutation, in complex with ARS1 DNA
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.42 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: S288c
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium acetateKOAc1
225 mMHEPES-KOHHEPES-KOH1
35 mMmagnesium acetateMgOAc21
41 mMBeta-mercptoethanolBeta-mercptoethanol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: C-flat-1.2/1.3
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 47170 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2EPU2.12image acquisition
4Gctf1.18CTF correction
7UCSF Chimera1.13.1model fitting
9PHENIX1.19-4092model refinement
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1387713
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 191281 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementHighest resolution: 2.98 Å

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