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- EMDB-75978: CryoEM structure of the human origin recognition complex with DNA... -

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Basic information

Entry
Database: EMDB / ID: EMD-75978
TitleCryoEM structure of the human origin recognition complex with DNA and CDC6 protein
Map data
Sample
  • Complex: Human ORC subunits (1-5) and Human CDC6 protein complex with 60bp DNA
    • Protein or peptide: Origin recognition complex subunit 2
    • Protein or peptide: Origin recognition complex subunit 4
    • DNA: DNA (29-MER)
    • DNA: DNA (29-MER)
    • Protein or peptide: Origin recognition complex subunit 1
    • Protein or peptide: Origin recognition complex subunit 3
    • Protein or peptide: Origin recognition complex subunit 5
    • Protein or peptide: Cell division control protein 6 homolog
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsOrigin Recognition Complex / ORC / origin licensing / WH domain / ATPase / REPLICATION / protein-DNA complex
Function / homology
Function and homology information


cellular response to vasopressin / traversing start control point of mitotic cell cycle / positive regulation of chromosome segregation / CDC6 association with the ORC:origin complex / polar body extrusion after meiotic divisions / origin recognition complex / E2F-enabled inhibition of pre-replication complex formation / inner kinetochore / nuclear origin of replication recognition complex / nuclear pre-replicative complex ...cellular response to vasopressin / traversing start control point of mitotic cell cycle / positive regulation of chromosome segregation / CDC6 association with the ORC:origin complex / polar body extrusion after meiotic divisions / origin recognition complex / E2F-enabled inhibition of pre-replication complex formation / inner kinetochore / nuclear origin of replication recognition complex / nuclear pre-replicative complex / DNA replication checkpoint signaling / DNA replication preinitiation complex / negative regulation of DNA replication / regulation of cyclin-dependent protein serine/threonine kinase activity / mitotic DNA replication checkpoint signaling / Transcription of E2F targets under negative control by DREAM complex / regulation of mitotic metaphase/anaphase transition / G1/S-Specific Transcription / spindle midzone / positive regulation of cytokinesis / cellular response to angiotensin / regulation of DNA replication / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / protein polymerization / Activation of ATR in response to replication stress / heterochromatin / positive regulation of fibroblast proliferation / Assembly of the ORC complex at the origin of replication / protein serine/threonine kinase binding / Assembly of the pre-replicative complex / CDK-mediated phosphorylation and removal of Cdc6 / Orc1 removal from chromatin / spindle pole / DNA replication / cell division / chromosome, telomeric region / protein-macromolecule adaptor activity / chromosome / nucleotide binding / negative regulation of cell population proliferation / centrosome / chromatin binding / nucleolus / chromatin / negative regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
Cell division protein Cdc6/18 / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3 insertion domain / : / Cdc6/ORC-like, ATPase lid domain / CDC6, C terminal / Orc1-like, AAA ATPase domain / AAA ATPase domain / Cdc6, C-terminal / CDC6, C terminal winged helix domain ...Cell division protein Cdc6/18 / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3 insertion domain / : / Cdc6/ORC-like, ATPase lid domain / CDC6, C terminal / Orc1-like, AAA ATPase domain / AAA ATPase domain / 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 / 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 5 / Origin recognition complex subunit 4 / Origin recognition complex subunit 1 / Origin recognition complex subunit 2 / DNA replication factor CDC6 / Origin recognition complex subunit 3
Similarity search - Component
Biological speciesHomo sapiens (human) / DNA molecule (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsChouhan OP / 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 10, 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_75978.png

Downloads & links

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Map

FileDownload / File: emd_75978.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 440 pix.
= 363.88 Å		0.83 Å/pix.
x 440 pix.
= 363.88 Å		0.83 Å/pix.
x 440 pix.
= 363.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.827 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.13439994 - 0.5572067
Average (Standard dev.)0.00029153717 (±0.011309713)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 363.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_75978_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_75978_half_map_2.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 : Human ORC subunits (1-5) and Human CDC6 protein complex with 60bp DNA

EntireName: Human ORC subunits (1-5) and Human CDC6 protein complex with 60bp DNA
Components
  • Complex: Human ORC subunits (1-5) and Human CDC6 protein complex with 60bp DNA
    • Protein or peptide: Origin recognition complex subunit 2
    • Protein or peptide: Origin recognition complex subunit 4
    • DNA: DNA (29-MER)
    • DNA: DNA (29-MER)
    • Protein or peptide: Origin recognition complex subunit 1
    • Protein or peptide: Origin recognition complex subunit 3
    • Protein or peptide: Origin recognition complex subunit 5
    • Protein or peptide: Cell division control protein 6 homolog
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Human ORC subunits (1-5) and Human CDC6 protein complex with 60bp DNA

SupramoleculeName: Human ORC subunits (1-5) and Human CDC6 protein complex with 60bp DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: Purified proteins were mixed with 60bp DNA in glycerol-free buffer containing ATP analogs and magnesium acetate. ORC:CDC6:DNA molar ratio of 1:2:3. Final protein concentration of 1 mg/mL, ...Details: Purified proteins were mixed with 60bp DNA in glycerol-free buffer containing ATP analogs and magnesium acetate. ORC:CDC6:DNA molar ratio of 1:2:3. Final protein concentration of 1 mg/mL, with 0.05% lauryl maltose neopentyl glycol (LMNG). Blotted using a Leica EM GP2 automatic plunge freezer.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 408 KDa

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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: Homo sapiens (human)
Molecular weightTheoretical: 66.063375 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSKPELKEDK MLEVHFVGDD DVLNHILDRE GGAKLKKERA QLLVNPKKII KKPEYDLEED DQEVLKDQNY VEIMGRDVQE SLKNGSATG GGNKVYSFQN RKHSEKMAKL ASELAKTPQK SVSFSLKNDP EITINVPQSS KGHSASDKVQ PKNNDKSEFL S TAPRSLRK ...String:
MSKPELKEDK MLEVHFVGDD DVLNHILDRE GGAKLKKERA QLLVNPKKII KKPEYDLEED DQEVLKDQNY VEIMGRDVQE SLKNGSATG GGNKVYSFQN RKHSEKMAKL ASELAKTPQK SVSFSLKNDP EITINVPQSS KGHSASDKVQ PKNNDKSEFL S TAPRSLRK RLIVPRSHSD SESEYSASNS EDDEGVAQEH EEDTNAVIFS QKIQAQNRVV SAPVGKETPS KRMKRDKTSD LV EEYFEAH SSSKVLTSDR TLQKLKRAKL DQQTLRNLLS KVSPSFSAEL KQLNQQYEKL FHKWMLQLHL GFNIVLYGLG SKR DLLERF RTTMLQDSIH VVINGFFPGI SVKSVLNSIT EEVLDHMGTF RSILDQLDWI VNKFKEDSSL ELFLLIHNLD SQML RGEKS QQIIGQLSSL HNIYLIASID HLNAPLMWDH AKQSLFNWLW YETTTYSPYT EETSYENSLL VKQSGSLPLS SLTHV LRSL TPNARGIFRL LIKYQLDNQD NPSYIGLSFQ DFYQQCREAF LVNSDLTLRA QLTEFRDHKL IRTKKGTDGV EYLLIP VDN GTLTDFLEKE EEEA

UniProtKB: Origin recognition complex subunit 2

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Macromolecule #2: Origin recognition complex subunit 4

MacromoleculeName: Origin recognition complex subunit 4 / type: protein_or_peptide / ID: 2 / Details: No Tag, Native protein / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50.443266 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSSRKSKSNS LIHTECLSQV QRILRERFCR QSPHSNLFGV QVQYKHLSEL LKRTALHGES NSVLIIGPRG SGKTMLINHA LKELMEIEE VSENVLQVHL NGLLQINDKI ALKEITRQLN LENVVGDKVF GSFAENLSFL LEALKKGDRT SSCPVIFILD E FDLFAHHK ...String:
MSSRKSKSNS LIHTECLSQV QRILRERFCR QSPHSNLFGV QVQYKHLSEL LKRTALHGES NSVLIIGPRG SGKTMLINHA LKELMEIEE VSENVLQVHL NGLLQINDKI ALKEITRQLN LENVVGDKVF GSFAENLSFL LEALKKGDRT SSCPVIFILD E FDLFAHHK NQTLLYNLFD ISQSAQTPIA VIGLTCRLDI LELLEKRVKS RFSHRQIHLM NSFGFPQYVK IFKEQLSLPA EF PDKVFAE KWNENVQYLS EDRSVQEVLQ KHFNISKNLR SLHMLLMLAL NRVTASHPFM TAVDLMEASQ LCSMDSKANI VHG LSVLEI CLIIAMKHLN DIYEEEPFNF QMVYNEFQKF VQRKAHSVYN FEKPVVMKAF EHLQQLELIK PMERTSGNSQ REYQ LMKLL LDNTQIMNAL QKYPNCPTDV RQWATSSLSW L

UniProtKB: Origin recognition complex subunit 4

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Macromolecule #5: Origin recognition complex subunit 1

MacromoleculeName: Origin recognition complex subunit 1 / type: protein_or_peptide / ID: 5
Details: N-terminal TwinStrep-SUMOstar-TEV tag, which was cleaved off using TEV protease, and G remained at the N-terminal after TEV cleavage.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 97.556914 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMAHYPTRLK TRKTYSWVGR PLLDRKLHYQ TYREMCVKTE GCSTEIHIQI GQFVLIEGDD DENPYVAKLL ELFEDDSDPP PKKRARVQW FVRFCEVPAC KRHLLGRKPG AQEIFWYDYP ACDSNINAET IIGLVRVIPL APKDVVPTNL KNEKTLFVKL S WNEKKFRP ...String:
GMAHYPTRLK TRKTYSWVGR PLLDRKLHYQ TYREMCVKTE GCSTEIHIQI GQFVLIEGDD DENPYVAKLL ELFEDDSDPP PKKRARVQW FVRFCEVPAC KRHLLGRKPG AQEIFWYDYP ACDSNINAET IIGLVRVIPL APKDVVPTNL KNEKTLFVKL S WNEKKFRP LSSELFAELN KPQESAAKCQ KPVRAKSKSA ESPSWTPAEH VAKRIESRHS ASKSRQTPTH PLTPRARKRL EL GNLGNPQ MSQQTSCASL DSPGRIKRKV AFSEITSPSK RSQPDKLQTL SPALKAPEKT RETGLSYTED DKKASPEHRI ILR TRIAAS KTIDIREERT LTPISGGQRS SVVPSVILKP ENIKKRDAKE AKAQNEATST PHRIRRKSSV LTMNRIRQQL RFLG NSKSD QEEKEILPAA EISDSSSDEE EASTPPLPRR APRTVSRNLR SSLKSSLHTL TKVPKKSLKP RTPRCAAPQI RSRSL AAQE PASVLEEARL RLHVSAVPES LPCREQEFQD IYNFVESKLL DHTGGCMYIS GVPGTGKTAT VHEVIRCLQQ AAQAND VPP FQYIEVNGMK LTEPHQVYVQ ILQKLTGQKA TANHAAELLA KQFCTRGSPQ ETTVLLVDEL DLLWTHKQDI MYNLFDW PT HKEARLVVLA IANTMDLPER IMMNRVSSRL GLTRMCFQPY TYSQLQQILR SRLKHLKAFE DDAIQLVARK VAALSGDA R RCLDICRRAT EICEFSQQKP DSPGLVTIAH SMEAVDEMFS SSYITAIKNS SVLEQSFLRA ILAEFRRSGL EEATFQQIY SQHVALCRME GLPYPTMSET MAVCSHLGSC RLLLVEPSRN DLLLRVRLNV SQDDVLYALK DE

UniProtKB: Origin recognition complex subunit 1

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Macromolecule #6: Origin recognition complex subunit 3

MacromoleculeName: Origin recognition complex subunit 3 / type: protein_or_peptide / ID: 6
Details: N-terminal TwinStrep-SUMOstar-TEV tag, which was cleaved off using TEV protease, and G remained at the N-terminal after TEV cleavage.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 82.422109 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMATSSMSKG CFVFKPNSKK RKISLPIEDY FNKGKNEPED SKLRFETYQL IWQQMKSENE RLQEELNKNL FDNLIEFLQK SHSGFQKNS RDLGGQIKLR EIPTAALVLG VNVTDHDLTF GSLTEALQNN VTPYVVSLQA KDCPDMKHFL QKLISQLMDC C VDIKSKEE ...String:
GMATSSMSKG CFVFKPNSKK RKISLPIEDY FNKGKNEPED SKLRFETYQL IWQQMKSENE RLQEELNKNL FDNLIEFLQK SHSGFQKNS RDLGGQIKLR EIPTAALVLG VNVTDHDLTF GSLTEALQNN VTPYVVSLQA KDCPDMKHFL QKLISQLMDC C VDIKSKEE ESVHVTQRKT HYSMDSLSSW YMTVTQKTDP KMLSKKRTTS SQWQSPPVVV ILKDMESFAT KVLQDFIIIS SQ HLHEFPL ILIFGIATSP IIIHRLLPHA VSSLLCIELF QSLSCKEHLT TVLDKLLLTT QFPFKINEKV LQVLTNIFLY HDF SVQNFI KGLQLSLLEH FYSQPLSVLC CNLPEAKRRI NFLSNNQCEN IRRLPSFRRY VEKQASEKQV ALLTNERYLK EETQ LLLEN LHVYHMNYFL VLRCLHKFTS SLPKYPLGRQ IRELYCTCLE KNIWDSEEYA SVLQLLRMLA KDELMTILEK CFKVF KSYC ENHLGSTAKR IEEFLAQFQS LDETKEEEDA SGSQPKGLQK TDLYHLQKSL LEMKELRRSK KQTKFEVLRE NVVNFI DCL VREYLLPPET QPLHEVVYFS AAHALREHLN AAPRIALHTA LNNPYYYLKN EALKSEEGCI PNIAPDICIA YKLHLEC SR LINLVDWSEA FATVVTAAEK MDANSATSEE MNEIIHARFI RAVSELELLG FIKPTKQKTD HVARLTWGGC

UniProtKB: Origin recognition complex subunit 3

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Macromolecule #7: Origin recognition complex subunit 5

MacromoleculeName: Origin recognition complex subunit 5 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50.349934 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MPHLENVVLC RESQVSILQS LFGERHHFSF PSIFIYGHTA SGKTYVTQTL LKTLELPHVF VNCVECFTLR LLLEQILNKL NHLSSSEDG CSTEITCETF NDFVRLFKQV TTAENLKDQT VYIVLDKAEY LRDMEANLLP GFLRLQELAD RNVTVLFLSE I VWEKFRPN ...String:
MPHLENVVLC RESQVSILQS LFGERHHFSF PSIFIYGHTA SGKTYVTQTL LKTLELPHVF VNCVECFTLR LLLEQILNKL NHLSSSEDG CSTEITCETF NDFVRLFKQV TTAENLKDQT VYIVLDKAEY LRDMEANLLP GFLRLQELAD RNVTVLFLSE I VWEKFRPN TGCFEPFVLY FPDYSIGNLQ KILSHDHPPE YSADFYAAYI NILLGVFYTV CRDLKELRHL AVLNFPKYCE PV VKGEASE RDTRKLWRNI EPHLKKAMQT VYLREISSSQ WEKLQKDDTD PGQLKGLSAH THVELPYYSK FILIAAYLAS YNP ARTDKR FFLKHHGKIK KTNFLKKHEK TSNHLLGPKP FPLDRLLAIL YSIVDSRVAP TANIFSQITS LVTLQLLTLV GHDD QLDGP KYKCTVSLDF IRAIARTVNF DIIKYLYDFL

UniProtKB: Origin recognition complex subunit 5

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Macromolecule #8: Cell division control protein 6 homolog

MacromoleculeName: Cell division control protein 6 homolog / type: protein_or_peptide / ID: 8
Details: N-terminal 8XHis-SUMOstar-TEV tag, which was cleaved off using TEV protease, and G remained at the N-terminal after TEV cleavage.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 62.877402 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GMPQTRSQAQ ATISFPKRKL SRALNKAKNS SDAKLEPTNV QTVTCSPRVK ALPLSPRKRL GDDNLCNTPH LPPCSPPKQG KKENGPPHS HTLKGRRLVF DNQLTIKSPS KRELAKVHQN KILSSVRKSQ EITTNSEQRC PLKKESACVR LFKQEGTCYQ Q AKLVLNTA ...String:
GMPQTRSQAQ ATISFPKRKL SRALNKAKNS SDAKLEPTNV QTVTCSPRVK ALPLSPRKRL GDDNLCNTPH LPPCSPPKQG KKENGPPHS HTLKGRRLVF DNQLTIKSPS KRELAKVHQN KILSSVRKSQ EITTNSEQRC PLKKESACVR LFKQEGTCYQ Q AKLVLNTA VPDRLPARER EMDVIRNFLR EHICGKKAGS LYLSGAPGTG KTACLSRILQ DLKKELKGFK TIMLNCMSLR TA QAVFPAI AQEICQEEVS RPAGKDMMRK LEKHMTAEKG PMIVLVLDEM DQLDSKGQDV LYTLFEWPWL SNSHLVLIGI ANT LDLTDR ILPRLQAREK CKPQLLNFPP YTRNQIVTIL QDRLNQVSRD QVLDNAAVQF CARKVSAVSG DVRKALDVCR RAIE IVESD VKSQTILKPL SECKSPSEPL IPKRVGLIHI SQVISEVDGN RMTLSQEGAQ DSFPLQQKIL VCSLMLLIRQ LKIKE VTLG KLYEAYSKVC RKQQVAAVDQ SECLSLSGLL EARGILGLKR NKETRLTKVF FKIEEKEIEH ALKDKALIGN ILATGL P

UniProtKB: DNA replication factor CDC6

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Macromolecule #3: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 18.630834 KDa
SequenceString: (DG)(DG)(DG)(DC)(DT)(DG)(DC)(DT)(DG)(DC) (DT)(DG)(DC)(DG)(DG)(DA)(DG)(DA)(DG)(DT) (DG)(DC)(DG)(DC)(DT)(DG)(DG)(DG)(DT) (DC)(DT)(DC)(DT)(DC)(DC)(DC)(DG)(DT)(DG) (DG) (DT)(DA)(DT)(DG)(DC)(DG) ...String:
(DG)(DG)(DG)(DC)(DT)(DG)(DC)(DT)(DG)(DC) (DT)(DG)(DC)(DG)(DG)(DA)(DG)(DA)(DG)(DT) (DG)(DC)(DG)(DC)(DT)(DG)(DG)(DG)(DT) (DC)(DT)(DC)(DT)(DC)(DC)(DC)(DG)(DT)(DG) (DG) (DT)(DA)(DT)(DG)(DC)(DG)(DC)(DG) (DC)(DG)(DG)(DG)(DG)(DA)(DC)(DC)(DA)(DT) (DA)(DC)

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Macromolecule #4: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 18.364727 KDa
SequenceString: (DG)(DT)(DA)(DT)(DG)(DG)(DT)(DC)(DC)(DC) (DC)(DG)(DC)(DG)(DC)(DG)(DC)(DA)(DT)(DA) (DC)(DC)(DA)(DC)(DG)(DG)(DG)(DA)(DG) (DA)(DG)(DA)(DC)(DC)(DC)(DA)(DG)(DC)(DG) (DC) (DA)(DC)(DT)(DC)(DT)(DC) ...String:
(DG)(DT)(DA)(DT)(DG)(DG)(DT)(DC)(DC)(DC) (DC)(DG)(DC)(DG)(DC)(DG)(DC)(DA)(DT)(DA) (DC)(DC)(DA)(DC)(DG)(DG)(DG)(DA)(DG) (DA)(DG)(DA)(DC)(DC)(DC)(DA)(DG)(DC)(DG) (DC) (DA)(DC)(DT)(DC)(DT)(DC)(DC)(DG) (DC)(DA)(DG)(DC)(DA)(DG)(DC)(DA)(DG)(DC) (DC)(DC)

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Macromolecule #9: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 9 / 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 #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / 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

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMKClPottesium chloride
25.0 mMC8H18N2O4SHEPES
2.0 mMC4H10O2S2DTT

Details: 25 mM HEPES pH 7.5, 100 mM KCl, 2 mM DTT, 0.05% LMNG
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
Details: The sample was applied to a glow-discharged Quantifoil R 0.6/1 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds, blotted for 3 seconds, and plunged into ...Details: The sample was applied to a glow-discharged Quantifoil R 0.6/1 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds, blotted for 3 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 KCl, 5 mM Mg(OAc)2, 2 mM DTT) for 20 minutes

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 7088 / 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, ...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 using ThermoFisher EPU data collection software.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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 selectionDetails: 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 correctionSoftware - Name: Warp (ver. 1.0.9)
Software - details: WARP did inital CTF estimation and correction to whole micrographs and particle stacks
Details: 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: The starting model was generated using the previous HsORC (7JPS) structure. Density map, which was initially generated using ab initio methods and subsequently underwent multiple refinements and reconstructions.
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Number images used: 130819
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)
Software - details: cryoSPARC heterogeneous refinement was used for initial angular assignment
Details: Multiple rounds of cryoSPARC heterogeneous refinement for generated ab initio maps.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)
Details: cryoSPARC non-uniform refinement was used for final angle assignment and refinement
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 4.7.1)
Software - details: Non-uniform refinement was carried out for the final reconstruction.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7jps


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial local fitting was done using Chimera, and then Coot was used for flexible fitting and model building.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-11rl:
CryoEM structure of the human origin recognition complex with DNA and CDC6 protein

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