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- PDB-7jgs: Structure of Drosophila ORC bound to poly(dA/dT) DNA and Cdc6 (co... -

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Basic information

Entry
Database: PDB / ID: 7jgs
TitleStructure of Drosophila ORC bound to poly(dA/dT) DNA and Cdc6 (conformation 2)
Components
  • (DNA (33-MER)) x 2
  • (Origin recognition complex subunit ...) x 5
  • AT22044p1
  • Cell division control protein
KeywordsREPLICATION / origin recognition complex / DNA replication initiation / DNA binding
Function / homology
Function and homology information


alpha-heterochromatin / larval feeding behavior / Assembly of the ORC complex at the origin of replication / CDC6 association with the ORC:origin complex / septin cytoskeleton / CDK-mediated phosphorylation and removal of Cdc6 / septin cytoskeleton organization / Activation of ATR in response to replication stress / Activation of the pre-replicative complex / eggshell chorion gene amplification ...alpha-heterochromatin / larval feeding behavior / Assembly of the ORC complex at the origin of replication / CDC6 association with the ORC:origin complex / septin cytoskeleton / CDK-mediated phosphorylation and removal of Cdc6 / septin cytoskeleton organization / Activation of ATR in response to replication stress / Activation of the pre-replicative complex / eggshell chorion gene amplification / Orc1 removal from chromatin / DNA amplification / origin recognition complex / positive regulation of border follicle cell migration / nuclear origin of replication recognition complex / olfactory learning / nuclear pre-replicative complex / DNA replication preinitiation complex / mitotic DNA replication checkpoint signaling / mitotic chromosome condensation / chromosome condensation / DNA replication origin binding / DNA replication initiation / heterochromatin / ribonucleoprotein complex binding / nuclear pore / GTPase activator activity / mitotic spindle organization / learning / DNA-templated DNA replication / mitotic cell cycle / DNA replication / learning or memory / cell division / chromatin binding / ATP hydrolysis activity / protein homodimerization activity / DNA binding / ATP binding / metal ion binding / nucleus / plasma membrane
Similarity search - Function
Origin recognition complex, subunit 6, metazoa/plant / Cell division protein Cdc6/18 / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3, N-terminal / Origin recognition complex (ORC) subunit 3 N-terminus / Origin recognition complex subunit 3 insertion domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cdc6, C-terminal / CDC6, C terminal ...Origin recognition complex, subunit 6, metazoa/plant / Cell division protein Cdc6/18 / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3, N-terminal / Origin recognition complex (ORC) subunit 3 N-terminus / Origin recognition complex subunit 3 insertion domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cdc6, C-terminal / CDC6, C terminal / CDC6, C terminal winged helix domain / AAA domain / 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 (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 / AAA ATPase domain / Origin recognition complex subunit 2 / Origin recognition complex, subunit 2 / AAA lid domain / AAA lid domain / Bromo adjacent homology (BAH) domain superfamily / Bromo adjacent homology domain / Bromo adjacent homology (BAH) domain / BAH domain / BAH domain profile. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Origin recognition complex subunit 1 / Origin recognition complex subunit 2 / Origin recognition complex subunit 5 / Origin recognition complex subunit 3 / Cell division control protein / Origin recognition complex subunit 4 / Origin recognition complex subunit 6
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSchmidt, J.M. / Bleichert, F.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-STG-757909 Switzerland
CitationJournal: Nat Commun / Year: 2020
Title: Structural mechanism for replication origin binding and remodeling by a metazoan origin recognition complex and its co-loader Cdc6.
Authors: Jan Marten Schmidt / Franziska Bleichert /
Abstract: Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2-7 replicative helicase onto replication origins. Here, we report cryo- ...Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2-7 replicative helicase onto replication origins. Here, we report cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc6. These structures reveal that Orc1 and Orc4 constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. A loop region near the catalytic Walker B motif of Orc1 directly contacts DNA, allosterically coupling DNA binding to ORC's ATPase site. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm2-7 loading in vitro. Together, these findings explain the distinct DNA sequence-dependencies of metazoan and S. cerevisiae initiators in origin recognition and support a model in which DNA geometry and bendability contribute to Mcm2-7 loading site selection in metazoans.
History
DepositionJul 19, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 9, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
B: Origin recognition complex subunit 2
C: AT22044p1
D: Origin recognition complex subunit 4
E: Origin recognition complex subunit 5
F: Origin recognition complex subunit 6
A: Origin recognition complex subunit 1
G: Cell division control protein
H: DNA (33-MER)
I: DNA (33-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)426,80217
Polymers424,6769
Non-polymers2,1268
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area44890 Å2
ΔGint-227 kcal/mol
Surface area112810 Å2

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Components

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Origin recognition complex subunit ... , 5 types, 5 molecules BDEFA

#1: Protein Origin recognition complex subunit 2 / / DmORC2


Mass: 69086.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Orc2, CG3041 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q24168
#3: Protein Origin recognition complex subunit 4 / / Orc4


Mass: 52277.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Orc4, Dmel\CG2917, DmORC4, dmOrc4, ORC, ORC4, orc4, rDmORC, CG2917, Dmel_CG2917
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9W102
#4: Protein Origin recognition complex subunit 5 /


Mass: 52175.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Orc5, CG7833 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q24169
#5: Protein Origin recognition complex subunit 6 /


Mass: 29284.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Orc6, CG1584 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9Y1B2
#6: Protein Origin recognition complex subunit 1 / / DmORC1


Mass: 54485.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Orc1, CG10667 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O16810

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Protein , 2 types, 2 molecules CG

#2: Protein AT22044p1 / FI24229p1 / GH28787p / LP02234p1 / Origin recognition complex subunit 3 / isoform A / isoform B


Mass: 82364.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Orc3, CG34315-RB, Dmel\CG4088, DmORC3, DmOrc3, l(2)49Fd, l(2)vr6, LAT, Lat, lat, lat-RA, latheo, ORC, ORC3, orc3, vr-6, vr6, CG4088, Dmel_CG4088
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q7K2L1
#7: Protein Cell division control protein / Cdc6


Mass: 48049.465 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Cdc6, CDC6, cdc6, DmCdc6, Dmel\CG5971, CG5971, Dmel_CG5971
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9VSM9

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DNA chain , 2 types, 2 molecules HI

#8: DNA chain DNA (33-MER)


Mass: 18206.602 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#9: DNA chain DNA (33-MER)


Mass: 18747.451 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 8 molecules

#10: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#11: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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: Drosophila ORC bound to poly(dA/dT) DNA duplex and Cdc6
Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES-KOH1
2250 mMpotassium acetate1
310 mMmagnesium acetate1
41 mMDTT1
51 mMATPAdenosine triphosphate1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Recombinantly expressed and purified Drosophila ORC and Cdc6 were mixed with DNA and the DNA-bound ternary complex purified by size exclusion chromatography.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / 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
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4GctfCTF correction
5RELIONCTF correction
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81248 / Symmetry type: POINT

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