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- EMDB-76021: Cryo-EM map of Yarrowia lipolytica ORC-Cdc6 bound to 54bp segment... -

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Basic information

Entry
Database: EMDB / ID: EMD-76021
TitleCryo-EM map of Yarrowia lipolytica ORC-Cdc6 bound to 54bp segment of OriC-061 DNA
Map dataCryo-EM unsharpened map of the Yarrowia lipolytica ORC-DNA-Cdc6 (ODC) complex with a 54bp dsDNA segment containing part of OriC-061.
Sample
  • Complex: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 54bp DNA segment of OriC-061
    • Protein or peptide: x 7 types
    • DNA: x 2 types
  • Ligand: x 4 types
KeywordsOrigin Recognition Complex / ORC / origin licensing / ATPase / REPLICATION
Function / homology
Function and homology information


nuclear DNA replication / nuclear origin of replication recognition complex / nuclear pre-replicative complex / DNA replication preinitiation complex / mitotic DNA replication checkpoint signaling / DNA replication origin binding / DNA replication initiation / DNA replication / protein-macromolecule adaptor activity / chromatin binding ...nuclear DNA replication / nuclear origin of replication recognition complex / nuclear pre-replicative complex / DNA replication preinitiation complex / mitotic DNA replication checkpoint signaling / DNA replication origin binding / DNA replication initiation / DNA replication / protein-macromolecule adaptor activity / chromatin binding / chromatin / ATP hydrolysis activity / DNA binding / ATP binding / nucleus
Similarity search - Function
Origin recognition complex, subunit 6, fungi / : / Cdc6/ORC-like, ATPase lid domain / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Orc1-like, AAA ATPase domain / AAA ATPase domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cdc6, C-terminal ...Origin recognition complex, subunit 6, fungi / : / Cdc6/ORC-like, ATPase lid domain / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Orc1-like, AAA ATPase domain / AAA ATPase domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cdc6, C-terminal / CDC6, C terminal winged helix 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 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 / : / : / Origin recognition complex subunit 2 RecA-like domain / ORC2 WHD / Origin recognition complex, subunit 2 / : / NACHT nucleoside triphosphatase / NACHT-NTPase domain profile. / 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-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
YALI0F31647p / YALI0F14773p / Origin recognition complex subunit 4 / Origin recognition complex subunit 2 / Origin recognition complex subunit 1 / YALI0C00671p / YALI0B01452p
Similarity search - Component
Biological speciesYarrowia lipolytica (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsBauer J / Joshua-Tor L
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: To Be Published
Title: Evolution of Origin Sequence and Recognition for Licensing of Eukaryotic DNA Replication
Authors: Bauer J / Zali N / Chouhan OP / El Demerdash O / Loell K / Kinney J / Joshua-Tor L / Stillman B
History
DepositionMar 11, 2026-
Header (metadata) releaseSep 30, 2026-
Map releaseSep 30, 2026-
UpdateSep 30, 2026-
Current statusSep 30, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_76021.png

Downloads & links

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Map

FileDownload / File: emd_76021.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM unsharpened map of the Yarrowia lipolytica ORC-DNA-Cdc6 (ODC) complex with a 54bp dsDNA segment containing part of OriC-061.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 440 pix.
= 382.36 Å		0.87 Å/pix.
x 440 pix.
= 382.36 Å		0.87 Å/pix.
x 440 pix.
= 382.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.869 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.28015724 - 0.8135828
Average (Standard dev.)0.00014268323 (±0.018574748)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 382.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Cryo-EM sharpened map of the Yarrowia lipolytica ORC-DNA-Cdc6...

Fileemd_76021_additional_1.map
AnnotationCryo-EM sharpened map of the Yarrowia lipolytica ORC-DNA-Cdc6 (ODC) complex with a 54bp dsDNA segment containing part of OriC-061. Used for model building.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map B of the Yarrowia lipolytica...

Fileemd_76021_half_map_1.map
AnnotationCryo-EM half map B of the Yarrowia lipolytica ORC-DNA-Cdc6 (ODC) complex with a 54bp dsDNA segment containing part of OriC-061.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map A of the Yarrowia lipolytica...

Fileemd_76021_half_map_2.map
AnnotationCryo-EM half map A of the Yarrowia lipolytica ORC-DNA-Cdc6 (ODC) complex with a 54bp dsDNA segment containing part of OriC-061.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ORC-Cdc6 complex of Yarrowia lipolytica bound to a 54bp DNA segme...

EntireName: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 54bp DNA segment of OriC-061
Components
  • Complex: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 54bp DNA segment of OriC-061
    • Protein or peptide: Origin recognition complex subunit 1
    • Protein or peptide: Origin recognition complex subunit 2
    • Protein or peptide: Origin recognition complex subunit 3 (Orc3)
    • Protein or peptide: Origin recognition complex subunit 4 (Orc4)
    • Protein or peptide: Origin recognition complex subunit 5 (Orc5)
    • Protein or peptide: Origin recognition complex subunit 6 (Orc6)
    • Protein or peptide: Cell division control protein 6 (Cdc6)
    • DNA: DNA (54-MER)
    • DNA: DNA (54-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: water

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Supramolecule #1: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 54bp DNA segme...

SupramoleculeName: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 54bp DNA segment of OriC-061
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Details: Purified YlORC1-6 was mixed with glycerol-free buffer containing ATP and magnesium acetate, a 54 bp OriC-061 fragment, and YlCdc6 at an ORC:DNA:Cdc6 molar ratio of 1:1.5:4 in a stepwise ...Details: Purified YlORC1-6 was mixed with glycerol-free buffer containing ATP and magnesium acetate, a 54 bp OriC-061 fragment, and YlCdc6 at an ORC:DNA:Cdc6 molar ratio of 1:1.5:4 in a stepwise fashion, followed by gel filtration. Final protein concentration of 1-1.25 mg/mL, with 0.05% lauryl maltose neopentyl glycol (LMNG). Blotted using a Leica EM GP2 automatic plunge freezer.
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 384 KDa

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Macromolecule #1: Origin recognition complex subunit 1

MacromoleculeName: Origin recognition complex subunit 1 / type: protein_or_peptide / ID: 1
Details: Contained N-terminal TwinStrep-SUMOstar-TEV tag which was left on to improve solubility of complex during concentration.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 96.564922 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KTGSLQDSEV NQEAKPEVKP EVKPETHINL KVSDGSSEIF FKIKKTTPLR RLMEAFAKR QGKEMDSLTF LYDGIEIQAD QTPEDLDMED NDIIEAHREQ IGGIPTTENL YFQGMTKEYT AIYSPEAKSA P KNKKKAAV ...String:
MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KTGSLQDSEV NQEAKPEVKP EVKPETHINL KVSDGSSEIF FKIKKTTPLR RLMEAFAKR QGKEMDSLTF LYDGIEIQAD QTPEDLDMED NDIIEAHREQ IGGIPTTENL YFQGMTKEYT AIYSPEAKSA P KNKKKAAV RAIRDQDNVE ISAGDVVLLK DDPDVEGKEF ALIQGLKHGD QGLEAKCVLM KLFNDAEALT PKHVIPNTNK NR YSKGQEL VMMNSIVDVL VEELHLPVNC YSFAEFEALS REDKKGDNVY FCRYVFDNDA NKTSVEFDWQ DITKDMCGFI DIL YELITD KPRKRRAAVK ASRQRSRHAR DEDESDFELE EEEEEEEEDD IEDIDEDDEY DSPVEQVKKA RTPKSAKKNT KKAP ATTPR KRALEDLDLP QPDHNTTPMT TPKKKRKTEN GHGLATPKRM FYKQALSDAT LPYKTADLSP SKLSPHQSAR AKLHV AAVP DTLPCRETEF SNVYLGIESA IRSGSGTCIF VSGTPGSGKT ATVREVVSQL QIRVEDNEIP DFLFVELNGM KLTNPH TTY ELLWEQLSGE RLAYNNAIKL LEHRFQQKSN DTPLVVVLDE LDQLVTLNQS VMYNFFNWPT LPHSKLIVVA IANTMDL PE RTLSNKISSR LGLTRIQFPG YTHEQLKLII ESRLGDIAES SGTVVRPDAI EFASRKIASV SGDARRALDL CRRAVEIA E LDSEEVQIKH IQQAANEATS TPIYNYLQGL PLAFKIFLCA LLARKRRNGL PSDSLGDIIE EIERMIKSSE NAGFLSHIL LQGGKRVRMA GFMNAVTELV EAGIIIQQSI KGERSAQVRL TIGVEEITSA LKNDDDVKGM L

UniProtKB: Origin recognition complex subunit 1

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Macromolecule #2: Origin recognition complex subunit 2

MacromoleculeName: Origin recognition complex subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 57.479059 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSRRKAPVSY SGLDTSDVSD EDFEVEDEVS TPKRRKTTSP RKQTPRRTAS PRKAPGTPSP VKRSLHDKSA RKKANRTLLD QSLGLVSED EDEIELAERI IGESRAPILD SSNDFHTDER SLANVGALIA AEDRVLFLDS SEGYFDQHKT RGRGNANTMA K APAIDHSV ...String:
MSRRKAPVSY SGLDTSDVSD EDFEVEDEVS TPKRRKTTSP RKQTPRRTAS PRKAPGTPSP VKRSLHDKSA RKKANRTLLD QSLGLVSED EDEIELAERI IGESRAPILD SSNDFHTDER SLANVGALIA AEDRVLFLDS SEGYFDQHKT RGRGNANTMA K APAIDHSV FFKYTNQANE LFHADQKKML RHAYRGMFSQ WIFELSEGFS LLFYGLGSKR ELLTDFVCEK VDSEIPILVI NG YNASVQF KSVLNSVVDV LYENHEDIFA KKGFVVRNKL PKDVDLLVKL VVDTMRDIEA GSKPSLVVLC HNVDGESLRI DKA STHLSQ LMSISQIWFV ASVDHIMAPL MWDSAKLASY NFVWHDVTTF APYTVETSFD DPLLLGKKAE AQGAKGVKYV LESV TPNHR SLYKNLIYCQ LEEFHNVADK RKLPEAEVGA LTGSTSISVD YDKVLTECLN ELTVSNKKDF QEKLKEFMDH KMVVA FDDK MGMKKLYIPF SKDVVQQILE GYLDA

UniProtKB: Origin recognition complex subunit 2

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Macromolecule #3: Origin recognition complex subunit 3 (Orc3)

MacromoleculeName: Origin recognition complex subunit 3 (Orc3) / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 78.749531 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSNHATLLQR EDQKTSYFLS DEIRALKRRR LDKSDPPKNG HKPEIDETND SGENSETAER EDEKTTLGEI ENEGHDNPDE NNPFARLLG GKEPSSGVNL RWNMYQEAWG AQKAKIDELL EMTNGNILEE IVEYVSESDA ENQIPAALVF PGSNIANHVR L FGQIREWL ...String:
MSNHATLLQR EDQKTSYFLS DEIRALKRRR LDKSDPPKNG HKPEIDETND SGENSETAER EDEKTTLGEI ENEGHDNPDE NNPFARLLG GKEPSSGVNL RWNMYQEAWG AQKAKIDELL EMTNGNILEE IVEYVSESDA ENQIPAALVF PGSNIANHVR L FGQIREWL GAVKGVHMVT LHARTCPSLK AVIKNIVSDL IESEEVAEEV EVREEDLNYD RRVKYDFSIL AEWCRKVGTD AS QRIVLIL EDVDSFDVKV LSNLVLMMHS YKDEIPFRLV FGIATSLEIF EHKMTKTSIR HLQGRVFDAQ ATSMFQSLFE NHM FNLNNK SIIVGPTILE DILKRQNVST ESIDAFISSL KYAYMSHYYS NPFSIFTSRL LDAGDEYEQI IDSNLTGEHI DALR MLPSF RALAESKTDA SEIDALLSDD SHIMDITKQA VHDFKVTARR VVSLINLFET IENVFGKFPM SWGKTEIYIP LVRGE LGES DFFKAVCESF KSQSDEKIQH LAQELAKDDL FDWLHDPDTI LDTITEALHN LKPFKQHLYH EIFVTDLATL QQNVFV PFQ RPAIETALAD PRHYLGIEDD DNKFKFVDPN ISTLFTLYRE SGIYINIYDW YVAFKECMPR SVIETELKKQ GLVPEEG ET VEDWDKRTLS WFYQAAAELK FIGCVRDTKR KVESVEKLIW RGL

UniProtKB: YALI0F14773p

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Macromolecule #4: Origin recognition complex subunit 4 (Orc4)

MacromoleculeName: Origin recognition complex subunit 4 (Orc4) / type: protein_or_peptide / ID: 4 / Details: Author provided reference is GenBank XP_504002.3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 57.684289 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MESEFPEIGG EITPTGDEMD VEIVEEVVEP AAPVLPPPQV PKPEAVSETM IDTIKSRTLS ILTGKSIPEP IFLDGEKARV YSLMENAIR FGEGNSCIIV GPRGTGKTLI VESALTELEE KYNSAGSQNN FITIRLSGYA QTDDKMAVRE IARQLDTVLL N QGQLIENK ...String:
MESEFPEIGG EITPTGDEMD VEIVEEVVEP AAPVLPPPQV PKPEAVSETM IDTIKSRTLS ILTGKSIPEP IFLDGEKARV YSLMENAIR FGEGNSCIIV GPRGTGKTLI VESALTELEE KYNSAGSQNN FITIRLSGYA QTDDKMAVRE IARQLDTVLL N QGQLIENK SISETLNQIL SLFDRADIDE SEKETVSLVF ILDEFDRFCS TTKQTLLYTL FDVAQSSRAP IAVIGLTPRI NA RELLEKR VRSRFSQRVV QVKRQHGMND FWAILRNAVI YPENLLTMVK EEGGNKTALH TDVDLDTVRY WNWHWESMFQ AGP LRDHVE RLFHTTKSCR EFFTSAILAV SQANPWINPN DFVTDVFERG VADTESFIEG LSDLELSLII CAAKVEVMFE VDQV NFNLA YEEYIKTAKE QREALRAVDL EGMATGTVAG FRIWSRGVAR AAWEKLESLN LLSPVEKSAK RVAKQLASDT SLDDE IRMT RVDVSLQELT NMLGNSHHLI QWTKIRR

UniProtKB: Origin recognition complex subunit 4

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Macromolecule #5: Origin recognition complex subunit 5 (Orc5)

MacromoleculeName: Origin recognition complex subunit 5 (Orc5) / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 52.939547 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MLPKDVVAAT RRQVSCRDTQ IKLLSVLLSE KAQEMPQSIL VHGEPSTGKS TVLKHLLKQS SINHSIILAE QCLTTRILLQ RTFRAVVED SGKTLADDFE IICENVTAFM ALLERFKAQY DFTKPHVIVL DGLDKLHENP SEIYHCFTRL NEMTSIRNVS F IFTISTLE ...String:
MLPKDVVAAT RRQVSCRDTQ IKLLSVLLSE KAQEMPQSIL VHGEPSTGKS TVLKHLLKQS SINHSIILAE QCLTTRILLQ RTFRAVVED SGKTLADDFE IICENVTAFM ALLERFKAQY DFTKPHVIVL DGLDKLHENP SEIYHCFTRL NEMTSIRNVS F IFTISTLE PRALITSSIP HVRFTRYTKE EVVTILSEHE LCRLPQTILS EAAKNGTEEE KDVLSRQFWG SYCQVLVDAL SP YASSDVS LYKQIARRIW PVYVDPVITG SADMRETAKL YVQSQHIFSS EFAVADSLVQ PGMEEALKRK RNNEQDLTGS YDL PLHSKY ILVAAYLASY NPERYDIRFF SKQKDGRKGR RDTGRRKRLT LNPRMLEAPP FELERMLAIL HSISPEEQFG TAAG VQSMS NIDLPGQIAT LTTLKLLVRT SGDPLDSRTK WKVNAGWGLI ERLARDIELP IHNYLLDENE

UniProtKB: YALI0B01452p

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Macromolecule #6: Origin recognition complex subunit 6 (Orc6)

MacromoleculeName: Origin recognition complex subunit 6 (Orc6) / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 41.218355 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNQQINAEIV KLVGNVPIPR EVTNLASTLM GKAQTIPMKP NETPARMALC AHVAIEKLLI ELQLPAPKQS QPPVPPRSYE KLLQLFREE LLGAPPGPST PRKRKSPMKN PELVAQRTPK TPRTARQVQK DIQESGRSDA NVIGEDLLNS EAIGEEDGLL P DTPSKTKK ...String:
MNQQINAEIV KLVGNVPIPR EVTNLASTLM GKAQTIPMKP NETPARMALC AHVAIEKLLI ELQLPAPKQS QPPVPPRSYE KLLQLFREE LLGAPPGPST PRKRKSPMKN PELVAQRTPK TPRTARQVQK DIQESGRSDA NVIGEDLLNS EAIGEEDGLL P DTPSKTKK SPQKSPRKGG PKQDDPQPAD IEFITKELRF PKHALEGVQR GFDFYWALVK DRWGLLFGLL MTIAFHIQHR SF TDTEATR EAFKQRALQL TRRAGMPEDR VEEWIGWTET ILKDQMWVKI LEQKSGIAPG VIQRQLDRQT SKSSFSGIGN MIP ASFAFN SYRKRNDYHN WKASMLVKMK ELKGQEGLEE SGTIKVQGE

UniProtKB: YALI0F31647p

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Macromolecule #7: Cell division control protein 6 (Cdc6)

MacromoleculeName: Cell division control protein 6 (Cdc6) / type: protein_or_peptide / ID: 7
Details: Originally contained an N-terminal 8xHis-TEV tag for purification, which was subsequently cleaved using TEV protease. A leftover glycine (residue 0) can be seen in the map. Chains K and L ...Details: Originally contained an N-terminal 8xHis-TEV tag for purification, which was subsequently cleaved using TEV protease. A leftover glycine (residue 0) can be seen in the map. Chains K and L are N-terminal regions of Cdc6.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 66.907148 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GMAVATPKRP QKRVRREPIR VPLKELEVNK GEDMGGQQVG EVDRGVSTEP LCLKRRKVEC FEDDSVRVLS PPRESCDPFT DSSATSGSS FASPPPLHPH LVELNKIKSM FSRGSKGHIL AADQEMVGRQ VEEATLLRYF EGRLQAKYSQ PGAALYVSGP P GTGKTALL ...String:
GMAVATPKRP QKRVRREPIR VPLKELEVNK GEDMGGQQVG EVDRGVSTEP LCLKRRKVEC FEDDSVRVLS PPRESCDPFT DSSATSGSS FASPPPLHPH LVELNKIKSM FSRGSKGHIL AADQEMVGRQ VEEATLLRYF EGRLQAKYSQ PGAALYVSGP P GTGKTALL QRVMDKVFRG KEGIKVASIN CMLAPSARAI MNLIYKQLSG VEENEALSAD ISFDKSVAKL EELFMCQTSK EF AERGTSI VVLDEIDHIM TRDQDILFRI FEWAFCKGSR LILVGIANAL DLTDRFLPRL KANNFYPQLL KFKPYDAVQI ASI IKSRIV KASDEFSREH SSLKKEVVVK KEEDLILSPL NTPKKTQIDP TTLTLTPPHT PTDKTPAVAP TTMAIHPAAI QLCA RKASA NTGDLRKAFD ICRKALEISE QEFIQKLAQN DPSTVSKPVV SIATMARVCS QVFGGNNSQR IKMLNLQQKA VLCTV ASAE KQLSIEAITS GVDVPLTIQR LFDHYTSSCK KHRMLSPLPF NEFLDVCSAL ESYSVINITG ICGKKNLGIN GKGRAS KGG TGASKGEVYG IRDDYVQRKV TLNVQRMDIA SAIEVEWLQK YL

UniProtKB: YALI0C00671p

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Macromolecule #8: DNA (54-MER)

MacromoleculeName: DNA (54-MER) / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 16.55168 KDa
SequenceString: (DT)(DG)(DG)(DT)(DA)(DC)(DC)(DG)(DA)(DT) (DC)(DC)(DC)(DA)(DA)(DT)(DA)(DT)(DT)(DA) (DC)(DA)(DC)(DC)(DC)(DA)(DA)(DG)(DT) (DA)(DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DA) (DA) (DG)(DC)(DT)(DA)(DA)(DA) ...String:
(DT)(DG)(DG)(DT)(DA)(DC)(DC)(DG)(DA)(DT) (DC)(DC)(DC)(DA)(DA)(DT)(DA)(DT)(DT)(DA) (DC)(DA)(DC)(DC)(DC)(DA)(DA)(DG)(DT) (DA)(DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DA) (DA) (DG)(DC)(DT)(DA)(DA)(DA)(DA)(DG) (DT)(DA)(DA)(DC)(DT)(DC)

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Macromolecule #9: DNA (54-MER)

MacromoleculeName: DNA (54-MER) / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Yarrowia lipolytica (yeast)
Molecular weightTheoretical: 16.719715 KDa
SequenceString: (DG)(DA)(DG)(DT)(DT)(DA)(DC)(DT)(DT)(DT) (DT)(DA)(DG)(DC)(DT)(DT)(DA)(DT)(DG)(DC) (DA)(DT)(DG)(DC)(DT)(DA)(DC)(DT)(DT) (DG)(DG)(DG)(DT)(DG)(DT)(DA)(DA)(DT)(DA) (DT) (DT)(DG)(DG)(DG)(DA)(DT) ...String:
(DG)(DA)(DG)(DT)(DT)(DA)(DC)(DT)(DT)(DT) (DT)(DA)(DG)(DC)(DT)(DT)(DA)(DT)(DG)(DC) (DA)(DT)(DG)(DC)(DT)(DA)(DC)(DT)(DT) (DG)(DG)(DG)(DT)(DG)(DT)(DA)(DA)(DT)(DA) (DT) (DT)(DG)(DG)(DG)(DA)(DT)(DC)(DG) (DG)(DT)(DA)(DC)(DC)(DA)

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #11: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 11 / Number of copies: 3 / 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 #12: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 12 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #13: water

MacromoleculeName: water / type: ligand / ID: 13 / Number of copies: 22 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration1.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
25.0 mMHEPES
100.0 mMsodium chlorideNaCl
1.0 mMdithiothreitol
0.05 %lauryl maltose neopentyl glycol

Details: 25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, 0.05% LMNG
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Details: ethyl acetate wash
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
Details: Sample was applied to a non-glow discharged Quantifoil R 1.2/1.3 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds, blotted for 2.9 seconds, and plunged ...Details: Sample was applied to a non-glow discharged Quantifoil R 1.2/1.3 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds, blotted for 2.9 seconds, and plunged into liquid ethane using a Leica Automatic Plunge Freezer EM GP2..
DetailsPre-incubated in assembly buffer (50 mM HEPES pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, 1 mM ATP, 1 mM DTT, 10% glycerol) for 10 minutes before size exclusion chromatography.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 3 / Number real images: 20341 / Average electron dose: 43.2 e/Å2
Details: Cryo-electron microscopy data were collected using an FEI/ThermoFisher Titan Krios TEM operating at 300 keV. A Gatan K3 direct electron detector equipped with a BioQuantum energy filter was ...Details: Cryo-electron microscopy data were collected using an FEI/ThermoFisher Titan Krios TEM operating at 300 keV. A Gatan K3 direct electron detector equipped with a BioQuantum energy filter was utilized to semi-automatically collect dose-fractionated movies with ThermoFisher EPU data collection software. For the YlODC54bpOriC-061 data collection, 30-frame movies were collected over three consecutive days, resulting in 9309, 8758, and 2274 exposures taken, respectively, at a dose rate of 1.44 e/A^2 per frame, totaling a cumulative dose of 43.2 e/A^2.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3805196
Details: Particle picking used a BoxNet pre-trained neural network implemented in TensorFlow, with a particle diameter of 180 angstrom and a threshold score of 0.5
CTF correctionDetails: CTF correction was done first in WARP during exposure/micrograph pre-processing, and re-corrected during the final refinements/reconstruction of the map in cryoSPARC
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Details: Starting map was generated from data from previous collection of complex using shorter DNA (45bp OriC-061 vs. 54bp), which started from an ab-initio generated map and underwent multiple ...Details: Starting map was generated from data from previous collection of complex using shorter DNA (45bp OriC-061 vs. 54bp), which started from an ab-initio generated map and underwent multiple refinements and reconstructions, which was then input into a heterogeneous refinement along with other ab-initio classes for the particles/data used in the generation of this map.
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7)
Details: Non-uniform refinement was used for the final reconstruction.
Number images used: 51599
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Details: cryoSPARC heterogeneous refinement of generated ab initio maps and an imported map from the 45bp OriC-061 ODC map refinement, as described in the startup model section.
Final angle assignmentType: MAXIMUM LIKELIHOOD
Details: cryoSPARC non-uniform refinement was used for final angle assignment and refinement
Final 3D classificationNumber classes: 2 / Avg.num./class: 60002
Details: Final 3D classification classes differed between whether or not Cdc6 was visible. Further cleanup on the chosen class was done by subsetting particles via per-particle scale.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainDetails
source_name: AlphaFold, initial_model_type: in silico model
source_name: Other, initial_model_type: in silico modelGeneric B-DNA used as template, generated in Coot.
DetailsAlphaFold 2 models for each subunit were docked into the density individually using the "fit to map" functionality in ChimeraX, then refined using Coot. The density for the DNA was sharp enough to allow us to discern purines and pyrimidines, allowing us to produce a generic DNA-B form model of the respective DNA sequence and manually rebuild it in Coot. PHENIX Real Space Refine functionality was used to further process and finalize the structure.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-11st:
Structure of Yarrowia lipolytica ORC-Cdc6 bound to 54bp segment of OriC-061 DNA

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