EMDB-76023: Cryo-EM map of Yarrowia lipolytica ORC-Cdc6 bound to 60bp segment...

Basic information

Entry

Database: EMDB / ID: EMD-76023

• Title: Cryo-EM map of Yarrowia lipolytica ORC-Cdc6 bound to 60bp segment of OriA-006 mutant CNNGGNR DNA
• Map data: Unsharpened final EM map

Sample

• Complex: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 60bp DNA segment of a mutant of OriA-006 containing the CNNGGNR mutation
  • Protein or peptide: x 7 types
  • DNA: x 2 types
• Ligand: x 3 types

• Keywords: Origin Recognition Complex / ORC / origin licensing / ATPase / REPLICATION

Function / homology

Function:

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

Domain/homology:

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

Component:

YALI0F31647p / YALI0F14773p / Origin recognition complex subunit 4 / Origin recognition complex subunit 2 / Origin recognition complex subunit 1 / YALI0C00671p / YALI0B01452p

• Biological species: Yarrowia lipolytica (yeast)
• Method: single particle reconstruction / cryo EM / Resolution: 2.56 Å
• Authors: Bauer J / Joshua-Tor L

Funding support

United States, 1 items

Organization	Grant number	Country
Howard Hughes Medical Institute (HHMI)		United States

• Citation: Journal: 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

Deposition	Mar 11, 2026	-
Header (metadata) release	Sep 30, 2026	-
Map release	Sep 30, 2026	-
Update	Sep 30, 2026	-
Current status	Sep 30, 2026	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_76023.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Unsharpened final EM map
• Voxel size: X=Y=Z: 0.827 Å

Density

Contour Level	By AUTHOR: 0.05
Minimum - Maximum	-0.114714526 - 0.33958712
Average (Standard dev.)	-0.00012569709 (±0.007570897)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 432 432 432 Spacing 432 432 432
Cell	A=B=C: 357.264 Å α=β=γ: 90.0 °

Supplemental data

Additional map: Sharpened map used in building of structure

• File: emd_76023_additional_1.map
• Annotation: Sharpened map used in building of structure

Half map: #2

• File: emd_76023_half_map_1.map

Half map: #1

• File: emd_76023_half_map_2.map

Sample components

Entire : ORC-Cdc6 complex of Yarrowia lipolytica bound to a 60bp DNA segme...

• Entire: Name: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 60bp DNA segment of a mutant of OriA-006 containing the CNNGGNR mutation

Components

• Complex: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 60bp DNA segment of a mutant of OriA-006 containing the CNNGGNR mutation
  • Protein or peptide: Origin recognition complex subunit 1 (Orc1)
  • Protein or peptide: Origin recognition complex subunit 2 (Orc2)
  • 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 (60-MER)
  • DNA: DNA (60-MER)
• Ligand: MAGNESIUM ION
• Ligand: ADENOSINE-5'-TRIPHOSPHATE
• Ligand: water

Supramolecule #1: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 60bp DNA segme...

• Supramolecule: Name: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 60bp DNA segment of a mutant of OriA-006 containing the CNNGGNR mutation; 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 60 bp mutant OriA-006 fragment, and YlCdc6 at an ORC:DNA:Cdc6 molar ratio of 1:1.5:6 in a stepwise fashion. Final protein concentration of 1.6 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 weight: Theoretical: 384 KDa

Macromolecule #1: Origin recognition complex subunit 1 (Orc1)

• Macromolecule: Name: Origin recognition complex subunit 1 (Orc1) / 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.,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 weight: Theoretical: 96.564922 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

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

Macromolecule #2: Origin recognition complex subunit 2 (Orc2)

• Macromolecule: Name: Origin recognition complex subunit 2 (Orc2) / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Yarrowia lipolytica (yeast)
• Molecular weight: Theoretical: 57.479059 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

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

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

• Macromolecule: Name: 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 weight: Theoretical: 78.749531 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

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

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

• Macromolecule: Name: Origin recognition complex subunit 4 (Orc4) / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Yarrowia lipolytica (yeast)
• Molecular weight: Theoretical: 57.684289 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

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

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

• Macromolecule: Name: 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 weight: Theoretical: 52.939547 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

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

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

• Macromolecule: Name: 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 weight: Theoretical: 41.218355 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

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

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

• Macromolecule: Name: 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. Chains K and L are N-terminal regions of Cdc6.; Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Yarrowia lipolytica (yeast)
• Molecular weight: Theoretical: 66.850094 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MAVATPKRPQ KRVRREPIRV PLKELEVNKG EDMGGQQVGE VDRGVSTEPL CLKRRKVECF EDDSVRVLSP PRESCDPFTD SSATSGSSF ASPPPLHPHL VELNKIKSMF SRGSKGHILA ADQEMVGRQV EEATLLRYFE GRLQAKYSQP GAALYVSGPP G TGKTALLQ RVMDKVFRGK EGIKVASINC MLAPSARAIM NLIYKQLSGV EENEALSADI SFDKSVAKLE ELFMCQTSKE FA ERGTSIV VLDEIDHIMT RDQDILFRIF EWAFCKGSRL ILVGIANALD LTDRFLPRLK ANNFYPQLLK FKPYDAVQIA SII KSRIVK ASDEFSREHS SLKKEVVVKK EEDLILSPLN TPKKTQIDPT TLTLTPPHTP TDKTPAVAPT TMAIHPAAIQ LCAR KASAN TGDLRKAFDI CRKALEISEQ EFIQKLAQND PSTVSKPVVS IATMARVCSQ VFGGNNSQRI KMLNLQQKAV LCTVA SAEK QLSIEAITSG VDVPLTIQRL FDHYTSSCKK HRMLSPLPFN EFLDVCSALE SYSVINITGI CGKKNLGING KGRASK GGT GASKGEVYGI RDDYVQRKVT LNVQRMDIAS AIEVEWLQKY L

UniProtKB: YALI0C00671p

Macromolecule #8: DNA (60-MER)

• Macromolecule: Name: DNA (60-MER) / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Yarrowia lipolytica (yeast)
• Molecular weight: Theoretical: 18.335758 KDa

Sequence

String:

(DC)(DT)(DC)(DC)(DA)(DC)(DC)(DC)(DA)(DA) (DT)(DA)(DT)(DG)(DC)(DC)(DC)(DC)(DT)(DG) (DG)(DA)(DA)(DT)(DC)(DC)(DA)(DG)(DC) (DT)(DC)(DC)(DT)(DA)(DC)(DA)(DA)(DG)(DT) (DC) (DG)(DG)(DG)(DG)(DT)(DT)(DG)(DA) (DG)(DA)(DC)(DT)(DG)(DC)(DA)(DC)(DC)(DA) (DA)(DA)

Macromolecule #9: DNA (60-MER)

• Macromolecule: Name: DNA (60-MER) / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Yarrowia lipolytica (yeast)
• Molecular weight: Theoretical: 18.650898 KDa

Sequence

String:

(DT)(DT)(DT)(DG)(DG)(DT)(DG)(DC)(DA)(DG) (DT)(DC)(DT)(DC)(DA)(DA)(DC)(DC)(DC)(DC) (DG)(DA)(DC)(DT)(DT)(DG)(DT)(DA)(DG) (DG)(DA)(DG)(DC)(DT)(DG)(DG)(DA)(DT)(DT) (DC) (DC)(DA)(DG)(DG)(DG)(DG)(DC)(DA) (DT)(DA)(DT)(DT)(DG)(DG)(DG)(DT)(DG)(DG) (DA)(DG)

Macromolecule #10: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 4 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Macromolecule #11: ADENOSINE-5'-TRIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 11 / Number of copies: 4 / Formula: ATP
• Molecular weight: Theoretical: 507.181 Da

Chemical component information

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

Macromolecule #12: water

• Macromolecule: Name: water / type: ligand / ID: 12 / Number of copies: 16 / Formula: HOH
• Molecular weight: Theoretical: 18.015 Da

Chemical component information

ChemComp-HOH:
WATER

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1.6 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Name	Formula
50.0 mM	HEPES
150.0 mM	potassium acetate	KOAc
10.0 mM	magnesium acetate	MgOAc
1.0 mM	adenosine triphosphate	ATP
1.0 mM	dithiothreitol	DTT
0.05 %	lauryl maltose neopentyl glycol

Details: 50 mM HEPES pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, 1 mM ATP, 1 mM DTT

• Grid: Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Details: ethyl acetate wash
• Vitrification: Cryogen 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 at 25C and 95% humidity, blotted for 2.7 seconds, and plunged into liquid ethane using a Leica Automatic Plunge Freezer EM GP2..
• Details: Pre-incubated in assembly buffer (50 mM HEPES pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, 1 mM ATP, 1 mM DTT) for 10 minutes.

Electron microscopy

• Microscope: TFS KRIOS
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8428 / Average electron dose: 54.8 e/Ų; 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. FYlODC60bpOri-A006-CNNGGNR data collection included 8428 exposures from a single session, with 40 frames per movie, a dose rate of 1.37 e/A^2 per frame, and a cumulative dose of 54.8 e/A^2.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 2710398 ; Details: 8,428 movies were imported into cryoSPARC and underwent patch motion correction and patch CTF corrections to generate corrected micrographs. Template picking of micrographs commenced using representative 2D class averages of the particles used in the final YlODC60bpOri-A006-WT refinement, resulting in 4,708,382 particles being picked. Micrographs and respective particles were then analyzed using the Micrograph Junk Detector job, and after exposure and particle curation resulted in 2,710,398 particles from 7,330 micrographs. Particles were then extracted with a box size of 432 px and Fourier cropped to 128 px.
• CTF correction: Software - Name: cryoSPARC (ver. 4.7.1); Software - details: cryoSPARC Patch CTF Refinement and final CTF refinement in the Non-Uniform refinement job provided final CTF estimation/corrections.; Details: Patch CTF correction was carried out in cryoSPARC using the default settings, and was optimized during the refinements/reconstruction of the map in cryoSPARC.; Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• Startup model: Type of model: OTHER; Details: The YlODC60bpOriA-006-WT map was imported into the project and heterogeneous refinement with eight ab-initio maps and the imported YlODC60bpOriA-006-WT map was carried out on the entire particle dataset.
• Final reconstruction: Number classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.56 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1); Details: Non-uniform refinement was used for the final reconstruction.; Number images used: 51222
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1); Software - details: cryoSPARC heterogeneous refinement was used for initial angular assignment; Details: cryoSPARC heterogeneous refinement of generated ab initio maps and an imported map of YlODC60bpOriA-006-WT, as described in the startup model section.
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1); Details: cryoSPARC non-uniform refinement was used for final angle assignment and refinement
• Final 3D classification: Number classes: 3 / Avg.num./class: 28918 / Software - Name: cryoSPARC (ver. 4.7.1); Software - details: Non-uniform refinement was carried out for the final reconstruction.; Details: Final 3D classification classes differed between the presence of Cdc6 and the conformation of Orc1-AAA.

Atomic model buiding 1

• Initial model: Chain - Source name: Other / Chain - Initial model type: experimental model; Details: The initial model came from the experimental structure of YlODC60bpOriA-006-WT
• Details: The YlODC60bpOriA-006-WT structure was docked into the map using ChimeraX and used as a starting point, with the DNA sequence altered at the mutated sites and manual refinement in Coot used to refine the model.
• Refinement: Space: REAL / Protocol: FLEXIBLE FIT

Output model

PDB-11sv:
Structure of Yarrowia lipolytica ORC-Cdc6 bound to 60bp segment of OriA-006 mutant CNNGGNR DNA