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- EMDB-22329: Structure of Drosophila ORC bound to DNA (84 bp) and Cdc6 -

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Basic information

Entry
Database: EMDB / ID: EMD-22329
TitleStructure of Drosophila ORC bound to DNA (84 bp) and Cdc6
Map dataunsharpened cryo-EM map
Sample
  • Complex: Drosophila ORC bound to AT-rich DNA (84 bp) and Cdc6
    • Protein or peptide: x 7 types
    • DNA: x 2 types
  • Ligand: x 2 types
Keywordsorigin recognition complex / DNA replication initiation / DNA binding / REPLICATION
Function / homology
Function and homology information


alpha-heterochromatin / larval feeding behavior / 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 ...alpha-heterochromatin / larval feeding behavior / 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 / positive regulation of border follicle cell migration / Assembly of the ORC complex at the origin of replication / origin recognition complex / 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 / ribonucleoprotein complex binding / heterochromatin / 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 / protein homodimerization activity / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / metal ion binding / plasma membrane
Similarity search - Function
Origin recognition complex, subunit 6, metazoa/plant / : / ORC6 second cyclin-like domain / Cell division protein Cdc6/18 / : / Cdc6/ORC-like, ATPase lid domain / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3 insertion domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) ...Origin recognition complex, subunit 6, metazoa/plant / : / ORC6 second cyclin-like domain / Cell division protein Cdc6/18 / : / Cdc6/ORC-like, ATPase lid domain / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3 insertion domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / CDC6, C terminal / 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 / Cdc6, C-terminal / : / CDC6, C terminal winged helix domain / Orc1-like, AAA ATPase domain / Origin recognition complex subunit 2 / AAA domain / 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 / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSchmidt JM / Bleichert F
Funding support Switzerland, 1 items
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, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0055
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0055
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jgr
  • Surface level: 0.0055
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22329.png

Downloads & links

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Map

FileDownload / File: emd_22329.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened cryo-EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 300 pix.
= 258. Å		0.86 Å/pix.
x 300 pix.
= 258. Å		0.86 Å/pix.
x 300 pix.
= 258. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0055 / Movie #1: 0.0055
Minimum - Maximum-0.007289032 - 0.022539213
Average (Standard dev.)0.0000137456955 (±0.0010496711)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 258.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z258.000258.000258.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0070.0230.000

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Supplemental data

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Additional map: sharpened, masked cryo-EM map, filtered to local resolution

Fileemd_22329_additional.map
Annotationsharpened, masked cryo-EM map, filtered to local resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map

Fileemd_22329_half_map_1.map
Annotationhalf-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map

Fileemd_22329_half_map_2.map
Annotationhalf-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Drosophila ORC bound to AT-rich DNA (84 bp) and Cdc6

EntireName: Drosophila ORC bound to AT-rich DNA (84 bp) and Cdc6
Components
  • Complex: Drosophila ORC bound to AT-rich DNA (84 bp) and Cdc6
    • Protein or peptide: Origin recognition complex subunit 2
    • Protein or peptide: AT22044p1
    • Protein or peptide: Origin recognition complex subunit 4
    • Protein or peptide: Origin recognition complex subunit 5
    • Protein or peptide: Origin recognition complex subunit 6
    • Protein or peptide: Origin recognition complex subunit 1
    • Protein or peptide: Cell division control protein
    • DNA: DNA (34-MER)
    • DNA: DNA (34-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Drosophila ORC bound to AT-rich DNA (84 bp) and Cdc6

SupramoleculeName: Drosophila ORC bound to AT-rich DNA (84 bp) and Cdc6 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Source (natural)Organism: Drosophila melanogaster (fruit fly)

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Macromolecule #1: Origin recognition complex subunit 2

MacromoleculeName: Origin recognition complex subunit 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 69.086445 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSASNKGGYK TPRKENLMSI ENLTNSEEES EDLNTAMVGN AVESQPKVTS RRSTRRPSPT KKYQAYQKES NGKGQEERIV VNYVEMSDE RSSDAEDQEE EESIEESENA ARPAAKDLHL IQSEYNVAGT SMFGFNTPKK RDAMALAALN ATPCTPKTPK T PRLGVKTP ...String:
MSASNKGGYK TPRKENLMSI ENLTNSEEES EDLNTAMVGN AVESQPKVTS RRSTRRPSPT KKYQAYQKES NGKGQEERIV VNYVEMSDE RSSDAEDQEE EESIEESENA ARPAAKDLHL IQSEYNVAGT SMFGFNTPKK RDAMALAALN ATPCTPKTPK T PRLGVKTP DTKRKKSMDQ PKTPAHVRTR VKKQIAKIVA DSDEDFSGDE SDFRPSDEES SSSSSSSDAG NSSDNDAADD EP KTPSRAR RAIVVPVLPK TPSAARLRQS ARAKKSNEFV PESDGYFHSH ASSKILTSDH TLDRLKNPRL AADRVFSLLS EIK TSAEHE GSINAIMEEY RSYFPKWMCI LNEGFNILLY GLGSKHQLLQ SFHREVLHKQ TVLVVNGFFP SLTIKDMLDS ITSD ILDAG ISPANPHEAV DMIEEEFALI PETHLFLIVH NLDGAMLRNV KAQAILSRLA RIPNIHLLAS IDHINTPLLW DQGKL CSFN FSWWDCTTML PYTNETAFEN SLLVQNSGEL ALSSMRSVFS SLTTNSRGIY MLIVKYQLKN KGNATYQGMP FRDLYS SCR EAFLVSSDLA LRAQLTEFLD HKLVKSKRSV DGSEQLTIPI DGALLQQFLE EQEKK

UniProtKB: Origin recognition complex subunit 2

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Macromolecule #2: AT22044p1

MacromoleculeName: AT22044p1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 82.364523 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDPTISVSKG CFVYKNGATR AGKKAASKRK RPAAESSSLL GKEVVQQPFY EEYRKAWNQI NDHIADLQHR SYARTLEQLV DFVVGQAER DTPDEVLPTA ALLTGINQPD HLSQFTALTQ RLHAQRAAMV CVLQSRDCAT LKAAVETLVF GLVEDNAEVE Q MEDEDEDE ...String:
MDPTISVSKG CFVYKNGATR AGKKAASKRK RPAAESSSLL GKEVVQQPFY EEYRKAWNQI NDHIADLQHR SYARTLEQLV DFVVGQAER DTPDEVLPTA ALLTGINQPD HLSQFTALTQ RLHAQRAAMV CVLQSRDCAT LKAAVETLVF GLVEDNAEVE Q MEDEDEDE DGAERDRKRL RRSQCTMKQL KSWYTNNFDS EQKRRQLVVI LPDFECFNAS VLQDLILILS AHCGSLPFVL VL GVATAMT AVHGTLPYHV SSKIRLRVFQ TQAAPTGLNE VLDKVLLSPK YAFHLSGKTF KFLTHIFLYY DFSIHGFIQG FKY CLMEHF FGGNAFALCT DYSKALGRIK QLTHEDMETI RRLPSFRPYV EQINDCKRII AVLTDDDYLK KKLPQLLRDC LLHF LLFRC SLEFLTELVG DLPRCPLGKL RRELYVNCLN RAIISTPEYK ECLQMLSFLS KDEFVAKVNR ALERTEQFLV EEIAP LELG EACTAVLRPK LEAIRLAVDE VVKATMATIT TTSPNETRQA TDHLTPVASR QELKDQLLQR SKEDKMRHQL NTPTTQ FGR ALQKTLQLIE TQIVQDHLRA LQDAPPIHEL FVFSDIATVR RNIIGAPRAA LHTALNNPHF YMQCKCCELQ DQSLLVG TL PDLSVVYKLH LECGRMINLF DWLQAFRSVV SDSDHEEVAQ EQIDPQIQAR FTRAVAELQF LGYIKMSKRK TDHATRLT W

UniProtKB: Origin recognition complex subunit 3

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Macromolecule #3: Origin recognition complex subunit 4

MacromoleculeName: Origin recognition complex subunit 4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 52.277109 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: SNAMPEADRE LVSIRRFLKE RLQRDYTTLR GYAKERSNVR LLLQRTAEMG ESNSLLLLGP RGSGKTTLIN SVLADLLPNK SFGENTLIV HLDGNLHTDD RVALKSITVQ MQLENAADGK VFGSFAENLA FLLQCLKAGG KHSKSVIFIL EEFDLFCAHH N QTLLYNLF ...String:
SNAMPEADRE LVSIRRFLKE RLQRDYTTLR GYAKERSNVR LLLQRTAEMG ESNSLLLLGP RGSGKTTLIN SVLADLLPNK SFGENTLIV HLDGNLHTDD RVALKSITVQ MQLENAADGK VFGSFAENLA FLLQCLKAGG KHSKSVIFIL EEFDLFCAHH N QTLLYNLF DVSQSAQAPI CVLGVTCRLD VIELLEKRVK SRFSHRQVFL FPSLRRFEDY VDLCRDLLSL PTGNSLLLAA EK IYNLQNI QSGALYFSRN HFDPGEYGFS PRLRDAWNKQ ICKVLATQQA RSTLQALHDF DISEAYLKNF LFRLVAHLRP QSP HITAEK MAAVGSQFEG DDKIELLCGL SVLELCLIIA IKHHSQIYDR DSFNFEIIYA RFSKFAKVST TMQAVERSIV LKAF EHLRI AELIMPLTGG AGGGVGKVQK EFEMHKLALT YSQIHHCMQR YQALPTEVAQ WAQSSLI

UniProtKB: Origin recognition complex subunit 4

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Macromolecule #4: Origin recognition complex subunit 5

MacromoleculeName: Origin recognition complex subunit 5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 52.175066 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MEAICSSLEP LFPCREAAIE TLGELIGDSS ETYPSAIYLF GHSGTGKTAL TRAFLKECGK RQNVRTAHLN AIECYTTKIM LEILLDSLA PDQGDALKVD NMLDFVEQLR RQAATRVEDQ GFLIAVDNAE RLRDMDANVL PVLLRLQELT NLNLCVILLS Q LPFEKFYN ...String:
MEAICSSLEP LFPCREAAIE TLGELIGDSS ETYPSAIYLF GHSGTGKTAL TRAFLKECGK RQNVRTAHLN AIECYTTKIM LEILLDSLA PDQGDALKVD NMLDFVEQLR RQAATRVEDQ GFLIAVDNAE RLRDMDANVL PVLLRLQELT NLNLCVILLS Q LPFEKFYN KTGLSEIVCL HLAQYNKAET QRILGSDFQQ VRNQLLEQFA QDKKRLEICQ EAVTEDFYNN YLNLFLSVFY KA CRDVPEL QLTARKCLST YLEPVLDGTV DATDISRLWR HIAGPLRSAL TQIYMRIEKP AEEVEDFTAI EDQSVRKLAQ SLE LPYYAK FLLIAAFLAS HNAAKQDKRL FVKHHGKQRK RMQTVNARAK TTEKMSTTLG PKSFSIDRLL AIFYAILEEK VGLT CNLLS QISTLVHLNL LSFVSGEQNI MEGSARLQCT IGLEFVLQIG KVVGFNVRQY LCDFM

UniProtKB: Origin recognition complex subunit 5

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Macromolecule #5: Origin recognition complex subunit 6

MacromoleculeName: Origin recognition complex subunit 6 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 29.284129 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MTTLIEQLIT KMGLREEPNV LEKTTELVRL LELRSTNVPL QINEYGKIVL CADLASCMIG IAFDKEQALK LSGLRKSQYL NNKRMFEKL LDLNKLASVN DICVQLGLNE VARKAEELMT LFKGVAATED MGTDTSHPQY ATMAVFQACR LLKKKVSKSK L MPFSNLRP ...String:
MTTLIEQLIT KMGLREEPNV LEKTTELVRL LELRSTNVPL QINEYGKIVL CADLASCMIG IAFDKEQALK LSGLRKSQYL NNKRMFEKL LDLNKLASVN DICVQLGLNE VARKAEELMT LFKGVAATED MGTDTSHPQY ATMAVFQACR LLKKKVSKSK L MPFSNLRP SQFQLLEQQW ERMIAKHHKE SKVPSSTDME GKLKENQNEN IKGHEAKKAH KPPPEDYEIW KARMLAKAQA KL KELEASQ SHMDSQLLEA

UniProtKB: Origin recognition complex subunit 6

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Macromolecule #6: Origin recognition complex subunit 1

MacromoleculeName: Origin recognition complex subunit 1 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 54.485633 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: SNAPRRSIHL SNIVEQRVFE DDEIISTPKR GRSKKTVQDN DEDYSPKKSV QKTPTRTRRS STTTKTATTP SKGITTATAT PMTPSQKMK KIRAGELSPS MQQRTDLPAK DSSKSELQLA REQLHVSVVP KSLPCREREF ENIYAFLEGK IQDQCGGCMY V SGVPGTGK ...String:
SNAPRRSIHL SNIVEQRVFE DDEIISTPKR GRSKKTVQDN DEDYSPKKSV QKTPTRTRRS STTTKTATTP SKGITTATAT PMTPSQKMK KIRAGELSPS MQQRTDLPAK DSSKSELQLA REQLHVSVVP KSLPCREREF ENIYAFLEGK IQDQCGGCMY V SGVPGTGK TATVTGVIRT LQRMAKQNEL PAFEYLEING MRLTEPRQAY VQIYKQLTGK TVSWEQAHAL LEKRFTTPAP RR VTTVLLV DELDILCNRR QDVVYNLLDW PTKSAAKLVV VTIANTMDLP ERLLMGKVTS RLGLTRLTFQ PYSHKQLQEI VTA RLGGSE TFKGEAVQLV ARKVAAVSGD ARRALDICRR ATEIADTAAV KCVTMLHVQQ ALAEMIASAK VQAIRNCSRM EQIF LQAIA AEVTRTGVEE TTFMGVYQQV ETIAAFMGVT FPPPGRALRL CSKLGAERLI ISEHSRNDLF QKILLNVSAD DIHYA LRVE EMVN

UniProtKB: Origin recognition complex subunit 1

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Macromolecule #7: Cell division control protein

MacromoleculeName: Cell division control protein / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 48.049465 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: SNANNLPSPS RNKYQNARRV LNSAETQNLP GRESQLQELR EFFSNHLESQ TSGSLYVSGQ PGTGKTACLS LLLRDPDFSK RLQRVYINC TSIASVGAVY KKLCTELQLK VSGRTERDHL EAIQRHLKTA KRMLLLVLDE IDQLCTSRQE VLYTIFEWPA L PGSRILLV ...String:
SNANNLPSPS RNKYQNARRV LNSAETQNLP GRESQLQELR EFFSNHLESQ TSGSLYVSGQ PGTGKTACLS LLLRDPDFSK RLQRVYINC TSIASVGAVY KKLCTELQLK VSGRTERDHL EAIQRHLKTA KRMLLLVLDE IDQLCTSRQE VLYTIFEWPA L PGSRILLV GIANSLDLTD RALMRLNARC ELKPRLMHFP PYSKQQIVEI FKSRLAEAEV LDVFPPVTLQ LLAAKVSAIS GD VRRALDI GRRVVEIAEQ QKRDGEKEFN MKALQLEGKD AVEAKEKQDT LKPVQVTQVA AVLNKVYGAS QNLEEDIEAS FPL QQKLML CTLVLMLRNE RNKDISMGRL HEVYRRVCAK RNILALDQAE FTGTVDLVET RGILRIMRKK EPRLHKVLLQ WDEE EVHAA LSDKQLIASI LSDTACLSK

UniProtKB: Cell division control protein

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Macromolecule #8: DNA (34-MER)

MacromoleculeName: DNA (34-MER) / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.820561 KDa
SequenceString: (DA)(DT)(DC)(DT)(DT)(DT)(DA)(DC)(DA)(DT) (DC)(DT)(DT)(DG)(DT)(DT)(DA)(DT)(DT)(DT) (DT)(DA)(DC)(DA)(DG)(DA)(DT)(DT)(DT) (DT)(DA)(DT)(DG)(DT)(DT)(DT)(DA)(DG)(DA) (DT) (DC)(DT)(DT)(DT)(DT)(DA) ...String:
(DA)(DT)(DC)(DT)(DT)(DT)(DA)(DC)(DA)(DT) (DC)(DT)(DT)(DG)(DT)(DT)(DA)(DT)(DT)(DT) (DT)(DA)(DC)(DA)(DG)(DA)(DT)(DT)(DT) (DT)(DA)(DT)(DG)(DT)(DT)(DT)(DA)(DG)(DA) (DT) (DC)(DT)(DT)(DT)(DT)(DA)(DT)(DG) (DC)(DT)(DT)(DG)(DC)(DT)(DT)(DT)(DT)(DC) (DA)(DA) (DA)(DA)(DG)(DG)(DC)(DC)(DT) (DG)(DC)(DA)(DG)(DG)(DC)(DA)(DA)(DG)(DT) (DG)(DC)(DA) (DC)(DA)(DA)(DA)

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Macromolecule #9: DNA (34-MER)

MacromoleculeName: DNA (34-MER) / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.977766 KDa
SequenceString: (DT)(DT)(DT)(DG)(DT)(DG)(DC)(DA)(DC)(DT) (DT)(DG)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DG) (DC)(DC)(DT)(DT)(DT)(DT)(DG)(DA)(DA) (DA)(DA)(DG)(DC)(DA)(DA)(DG)(DC)(DA)(DT) (DA) (DA)(DA)(DA)(DG)(DA)(DT) ...String:
(DT)(DT)(DT)(DG)(DT)(DG)(DC)(DA)(DC)(DT) (DT)(DG)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DG) (DC)(DC)(DT)(DT)(DT)(DT)(DG)(DA)(DA) (DA)(DA)(DG)(DC)(DA)(DA)(DG)(DC)(DA)(DT) (DA) (DA)(DA)(DA)(DG)(DA)(DT)(DC)(DT) (DA)(DA)(DA)(DC)(DA)(DT)(DA)(DA)(DA)(DA) (DT)(DC) (DT)(DG)(DT)(DA)(DA)(DA)(DA) (DT)(DA)(DA)(DC)(DA)(DA)(DG)(DA)(DT)(DG) (DT)(DA)(DA) (DA)(DG)(DA)(DT)

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Macromolecule #10: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 4 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #11: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 11 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 7.6
Component:
ConcentrationName
25.0 mMHEPES-KOH
250.0 mMpotassium acetate
10.0 mMmagnesium acetate
1.0 mMDTT
1.0 mMATP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsRecombinantly expressed and purified Drosophila ORC and Cdc6 were mixed with DNA and the DNA-bound ternary complex purified by size exclusion chromatography.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 44520
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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