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- EMDB-13211: Structure of a DNA-loaded MCM double hexamer engaged with the Dbf... -

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Basic information

Entry
Database: EMDB / ID: EMD-13211
TitleStructure of a DNA-loaded MCM double hexamer engaged with the Dbf4-dependent kinase
Map dataAuto-refined structure of the DNA-bound MCM double hexamer engaged with DDK
Sample
  • Complex: DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-dependent kinase
    • Protein or peptide: x 8 types
    • DNA: x 2 types
  • Ligand: x 4 types
Function / homology
Function and homology information


positive regulation of spindle attachment to meiosis I kinetochore / positive regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / positive regulation of DNA replication initiation / positive regulation of kinetochore assembly / positive regulation of meiotic DNA double-strand break formation / negative regulation of exit from mitosis / Dbf4-dependent protein kinase complex / positive regulation of meiosis I / regulation of cell cycle phase transition / positive regulation of nuclear cell cycle DNA replication ...positive regulation of spindle attachment to meiosis I kinetochore / positive regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / positive regulation of DNA replication initiation / positive regulation of kinetochore assembly / positive regulation of meiotic DNA double-strand break formation / negative regulation of exit from mitosis / Dbf4-dependent protein kinase complex / positive regulation of meiosis I / regulation of cell cycle phase transition / positive regulation of nuclear cell cycle DNA replication / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / MCM complex / DNA replication preinitiation complex / double-strand break repair via break-induced replication / protein-containing complex localization / single-stranded DNA helicase activity / mitotic DNA replication checkpoint signaling / replication fork protection complex / mitotic DNA replication initiation / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / subtelomeric heterochromatin formation / chromosome, centromeric region / DNA replication initiation / heterochromatin formation / DNA helicase activity / protein serine/threonine kinase activator activity / helicase activity / chromosome segregation / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / non-specific serine/threonine protein kinase / positive regulation of protein phosphorylation / cell division / phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / DNA damage response / chromatin binding / chromatin / signal transduction / ATP hydrolysis activity / zinc ion binding / nucleoplasm / ATP binding / identical protein binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Regulatory subunit Dfp1/Him1, central region / Dfp1/Him1, central region / Zinc finger, DBF-type / DBF-type zinc finger superfamily / DBF zinc finger / Zinc finger DBF4-type profile. / Zinc finger in DBF-like proteins / MCM4, winged helix domain / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm3 ...Regulatory subunit Dfp1/Him1, central region / Dfp1/Him1, central region / Zinc finger, DBF-type / DBF-type zinc finger superfamily / DBF zinc finger / Zinc finger DBF4-type profile. / Zinc finger in DBF-like proteins / MCM4, winged helix domain / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / BRCT domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Winged helix-like DNA-binding domain superfamily / Protein kinase domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Cell division control protein 7 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DDK kinase regulatory subunit DBF4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGreiwe JF / Locke J / Nans A / Costa A
Funding supportEuropean Union, 4 items
OrganizationGrant numberCountry
Wellcome TrustFC001065
Medical Research Council (MRC, United Kingdom)FC001065
Cancer Research UKFC001065
European Research Council (ERC)820102European Union
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.
Authors: Julia F Greiwe / Thomas C R Miller / Julia Locke / Fabrizio Martino / Steven Howell / Anne Schreiber / Andrea Nans / John F X Diffley / Alessandro Costa /
Abstract: Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM ...Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.
History
DepositionJul 15, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0043
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0043
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7p5z
  • Surface level: 0.0043
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7p5z
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13211.png

Downloads & links

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Map

FileDownload / File: emd_13211.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAuto-refined structure of the DNA-bound MCM double hexamer engaged with DDK
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.0043 / Movie #1: 0.0043
Minimum - Maximum-0.01779138 - 0.030778106
Average (Standard dev.)1.2757343e-05 (±0.00096976425)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 518.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z518.400518.400518.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.0180.0310.000

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Supplemental data

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Mask #1

Fileemd_13211_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Histogram (log scale)

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Additional map: Sharpened map

Fileemd_13211_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Additional map: LAFTER filtered map, which was used for docking...

Fileemd_13211_additional_2.map
AnnotationLAFTER filtered map, which was used for docking of the Dbf4 BRCT domain.
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_13211_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_13211_half_map_2.map
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Sample components

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Entire : DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-depen...

EntireName: DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-dependent kinase
Components
  • Complex: DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-dependent kinase
    • Protein or peptide: DNA replication licensing factor MCM2
    • Protein or peptide: DNA replication licensing factor MCM3
    • Protein or peptide: DNA replication licensing factor MCM4
    • Protein or peptide: Minichromosome maintenance protein 5
    • Protein or peptide: DNA replication licensing factor MCM6
    • Protein or peptide: DNA replication licensing factor MCM7
    • Protein or peptide: Cell division control protein 7
    • Protein or peptide: DDK kinase regulatory subunit DBF4
    • DNA: DNA (53-MER)
    • DNA: DNA (53-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-depen...

SupramoleculeName: DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-dependent kinase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)

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Macromolecule #1: DNA replication licensing factor MCM2

MacromoleculeName: DNA replication licensing factor MCM2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 98.911539 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MSDNRRRRRE EDDSDSENEL PPSSPQQHFR GGMNPVSSPI GSPDMINPEG DDNEVDDVPD IDEVEEQMNE VDLMDDNMYE DYAADHNRD RYDPDQVDDR EQQELSLSER RRIDAQLNER DRLLRNVAYI DDEDEEQEGA AQLDEMGLPV QRRRRRRQYE D LENSDDDL ...String:
MSDNRRRRRE EDDSDSENEL PPSSPQQHFR GGMNPVSSPI GSPDMINPEG DDNEVDDVPD IDEVEEQMNE VDLMDDNMYE DYAADHNRD RYDPDQVDDR EQQELSLSER RRIDAQLNER DRLLRNVAYI DDEDEEQEGA AQLDEMGLPV QRRRRRRQYE D LENSDDDL LSDMDIDPLR EELTLESLSN VKANSYSEWI TQPNVSRTIA RELKSFLLEY TDETGRSVYG ARIRTLGEMN SE SLEVNYR HLAESKAILA LFLAKCPEEM LKIFDLVAME ATELHYPDYA RIHSEIHVRI SDFPTIYSLR ELRESNLSSL VRV TGVVTR RTGVFPQLKY VKFNCLKCGS ILGPFFQDSN EEIRISFCTN CKSKGPFRVN GEKTVYRNYQ RVTLQEAPGT VPPG RLPRH REVILLADLV DVSKPGEEVE VTGIYKNNYD GNLNAKNGFP VFATIIEANS IKRREGNTAN EGEEGLDVFS WTEEE EREF RKISRDRGII DKIISSMAPS IYGHRDIKTA VACSLFGGVP KNVNGKHSIR GDINVLLLGD PGTAKSQILK YVEKTA HRA VFATGQGASA VGLTASVRKD PITKEWTLEG GALVLADKGV CLIDEFDKMN DQDRTSIHEA MEQQSISISK AGIVTTL QA RCSIIAAANP NGGRYNSTLP LAQNVSLTEP ILSRFDILCV VRDLVDEEAD ERLATFVVDS HVRSHPENDE DREGEELK N NGESAIEQGE DEINEQLNAR QRRLQRQRKK EEEISPIPQE LLMKYIHYAR TKIYPKLHQM DMDKVSRVYA DLRRESIST GSFPITVRHL ESILRIAESF AKMRLSEFVS SYDLDRAIKV VVDSFVDAQK VSVRRQLRRS FAIYTLGH

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Macromolecule #2: DNA replication licensing factor MCM3

MacromoleculeName: DNA replication licensing factor MCM3 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 111.720242 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MKRRWKKNFI AVSAANRFKK ISSSGALENL YFQGEMEGST GFDGDATTFF APDAVFGDRV RRFQEFLDTF TSYRDSVRSI QVYNSNNAA NYNDDQDDAD ERDLLGDDDG DDLEKEKKAA SSTSLNILPH RIIISLDDLR EFDRSFWSGI LVEPAYFIPP A EKALTDLA ...String:
MKRRWKKNFI AVSAANRFKK ISSSGALENL YFQGEMEGST GFDGDATTFF APDAVFGDRV RRFQEFLDTF TSYRDSVRSI QVYNSNNAA NYNDDQDDAD ERDLLGDDDG DDLEKEKKAA SSTSLNILPH RIIISLDDLR EFDRSFWSGI LVEPAYFIPP A EKALTDLA DSMDDVPHPN ASAVSSRHPW KLSFKGSFGA HALSPRTLTA QHLNKLVSVE GIVTKTSLVR PKLIRSVHYA AK TGRFHYR DYTDATTTLT TRIPTPAIYP TEDTEGNKLT TEYGYSTFID HQRITVQEMP EMAPAGQLPR SIDVILDDDL VDK TKPGDR VNVVGVFKSL GAGGMNQSNS NTLIGFKTLI LGNTVYPLHA RSTGVAARQM LTDFDIRNIN KLSKKKDIFD ILSQ SLAPS IYGHDHIKKA ILLMLMGGVE KNLENGSHLR GDINILMVGD PSTAKSQLLR FVLNTASLAI ATTGRGSSGV GLTAA VTTD RETGERRLEA GAMVLADRGV VCIDEFDKMT DVDRVAIHEV MEQQTVTIAK AGIHTTLNAR CSVIAAANPV FGQYDV NRD PHQNIALPDS LLSRFDLLFV VTDDINEIRD RSISEHVLRT HRYLPPGYLE GEPVRERLNL SLAVGEDADI NPEEHSN SG AGVENEGEDD EDHVFEKFNP LLQAGAKLAK NKGNYNGTEI PKLVTIPFLR KYVQYAKERV IPQLTQEAIN VIVKNYTD L RNDDNTKKSP ITARTLETLI RLATAHAKVR LSKTVNKVDA KVAANLLRFA LLGEDIGNDI DEEESEYEEA LSKRSPQKS PKKRQRVRQP ASNSGSPIKS TPRRSTASSV NATPSSARRI LRFQDDEQNA GEDDNDIMSP LPADEEAELQ RRLQLGLRVS PRRREHLHA PEEGSSGPLT EVGTPRLPNV SSAGQDDEQQ QSVISFDNVE PGTISTGRLS LISGIIARLM QTEIFEEESY P VASLFERI NEELPEEEKF SAQEYLAGLK IMSDRNNLMV ADDKVWRV

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Macromolecule #3: DNA replication licensing factor MCM4

MacromoleculeName: DNA replication licensing factor MCM4 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 105.138375 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MSQQSSSPTK EDNNSSSPVV PNPDSVPPQL SSPALFYSSS SSQGDIYGRN NSQNLSQGEG NIRAAIGSSP LNFPSSSQRQ NSDVFQSQG RQGRIRSSAS ASGRSRYHSD LRSDRALPTS SSSLGRNGQN RVHMRRNDIH TSDLSSPRRI VDFDTRSGVN T LDTSSSSA ...String:
MSQQSSSPTK EDNNSSSPVV PNPDSVPPQL SSPALFYSSS SSQGDIYGRN NSQNLSQGEG NIRAAIGSSP LNFPSSSQRQ NSDVFQSQG RQGRIRSSAS ASGRSRYHSD LRSDRALPTS SSSLGRNGQN RVHMRRNDIH TSDLSSPRRI VDFDTRSGVN T LDTSSSSA PPSEASEPLR IIWGTNVSIQ ECTTNFRNFL MSFKYKFRKI LDEREEFINN TTDEELYYIK QLNEMRELGT SN LNLDARN LLAYKQTEDL YHQLLNYPQE VISIMDQTIK DCMVSLIVDN NLDYDLDEIE TKFYKVRPYN VGSCKGMREL NPN DIDKLI NLKGLVLRST PVIPDMKVAF FKCNVCDHTM AVEIDRGVIQ EPARCERIDC NEPNSMSLIH NRCSFADKQV IKLQ ETPDF VPDGQTPHSI SLCVYDELVD SCRAGDRIEV TGTFRSIPIR ANSRQRVLKS LYKTYVDVVH VKKVSDKRLD VDTST IEQE LMQNKVDHNE VEEVRQITDQ DLAKIREVAA REDLYSLLAR SIAPSIYELE DVKKGILLQL FGGTNKTFTK GGRYRG DIN ILLCGDPSTS KSQILQYVHK ITPRGVYTSG KGSSAVGLTA YITRDVDTKQ LVLESGALVL SDGGVCCIDE FDKMSDS TR SVLHEVMEQQ TISIAKAGII TTLNARSSIL ASANPIGSRY NPNLPVTENI DLPPPLLSRF DLVYLVLDKV DEKNDREL A KHLTNLYLED KPEHISQDDV LPVEFLTMYI SYAKEHIHPI ITEAAKTELV RAYVGMRKMG DDSRSDEKRI TATTRQLES MIRLAEAHAK MKLKNVVELE DVQEAVRLIR SAIKDYATDP KTGKIDMNLV QTGKSVIQRK LQEDLSREIM NVLKDQASDS MSFNELIKQ INEHSQDRVE SSDIQEALSR LQQEDKVIVL GEGVRRSVRL NNRV

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Macromolecule #4: Minichromosome maintenance protein 5

MacromoleculeName: Minichromosome maintenance protein 5 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 86.505734 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MSFDRPEIYS APVLQGESPN DDDNTEIIKS FKNFILEFRL DSQFIYRDQL RNNILVKNYS LTVNMEHLIG YNEDIYKKLS DEPSDIIPL FETAITQVAK RISILSRAQS ANNNDKDPEN TSMDTDSLLL NSLPTFQLIL NSNANQIPLR DLDSEHVSKI V RLSGIIIS ...String:
MSFDRPEIYS APVLQGESPN DDDNTEIIKS FKNFILEFRL DSQFIYRDQL RNNILVKNYS LTVNMEHLIG YNEDIYKKLS DEPSDIIPL FETAITQVAK RISILSRAQS ANNNDKDPEN TSMDTDSLLL NSLPTFQLIL NSNANQIPLR DLDSEHVSKI V RLSGIIIS TSVLSSRATY LSIMCRNCRH TTSITINNFN SITGNTVSLP RSCLSTIESE SSMANESNIG DESTKKNCGP DP YIIIHES SKFIDQQFLK LQEIPELVPV GEMPRNLTMT CDRYLTNKVI PGTRVTIVGI YSIYNSKNGA GSGRSGGGNG GSG VAIRTP YIKILGIQSD VETSSIWNSV TMFTEEEEEE FLQLSRNPKL YEILTNSIAP SIFGNEDIKK AIVCLLMGGS KKIL PDGMR LRGDINVLLL GDPGTAKSQL LKFVEKVSPI AVYTSGKGSS AAGLTASVQR DPMTREFYLE GGAMVLADGG VVCID EFDK MRDEDRVAIH EAMEQQTISI AKAGITTVLN SRTSVLAAAN PIYGRYDDLK SPGDNIDFQT TILSRFDMIF IVKDDH NEE RDISIANHVI NIHTGNANAM QNQQEENGSE ISIEKMKRYI TYCRLKCAPR LSPQAAEKLS SNFVTIRKQL LINELES TE RSSIPITIRQ LEAIIRITES LAKLELSPIA QERHVDEAIR LFQASTMDAA SQDPIGGLNQ ASGTSLSEIR RFEQELKR R LPIGWSTSYQ TLRREFVDTH RFSQLALDKA LYALEKHETI QLRHQGQNIY RSGV

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Macromolecule #5: DNA replication licensing factor MCM6

MacromoleculeName: DNA replication licensing factor MCM6 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 113.110211 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MSSPFPADTP SSNRPSNSSP PPSSIGAGFG SSSGLDSQIG SRLHFPSSSQ PHVSNSQTGP FVNDSTQFSS QRLQTDGSAT NDMEGNEPA RSFKSRALNH VKKVDDVTGE KVREAFEQFL EDFSVQSTDT GEVEKVYRAQ IEFMKIYDLN TIYIDYQHLS M RENGALAM ...String:
MSSPFPADTP SSNRPSNSSP PPSSIGAGFG SSSGLDSQIG SRLHFPSSSQ PHVSNSQTGP FVNDSTQFSS QRLQTDGSAT NDMEGNEPA RSFKSRALNH VKKVDDVTGE KVREAFEQFL EDFSVQSTDT GEVEKVYRAQ IEFMKIYDLN TIYIDYQHLS M RENGALAM AISEQYYRFL PFLQKGLRRV VRKYAPELLN TSDSLKRSEG DEGQADEDEQ QDDDMNGSSL PRDSGSSAAP GN GTSAMAT RSITTSTSPE QTERVFQISF FNLPTVHRIR DIRSEKIGSL LSISGTVTRT SEVRPELYKA SFTCDMCRAI VDN VEQSFK YTEPTFCPNP SCENRAFWTL NVTRSRFLDW QKVRIQENAN EIPTGSMPRT LDVILRGDSV ERAKPGDRCK FTGV EIVVP DVTQLGLPGV KPSSTLDTRG ISKTTEGLNS GVTGLRSLGV RDLTYKISFL ACHVISIGSN IGASSPDANS NNRET ELQM AANLQANNVY QDNERDQEVF LNSLSSDEIN ELKEMVKDEH IYDKLVRSIA PAVFGHEAVK KGILLQMLGG VHKSTV EGI KLRGDINICV VGDPSTSKSQ FLKYVVGFAP RSVYTSGKAS SAAGLTAAVV RDEEGGDYTI EAGALMLADN GICCIDE FD KMDISDQVAI HEAMEQQTIS IAKAGIHATL NARTSILAAA NPVGGRYNRK LSLRGNLNMT APIMSRFDLF FVILDDCN E KIDTELASHI VDLHMKRDEA IEPPFSAEQL RRYIKYARTF KPILTKEARS YLVEKYKELR KDDAQGFSRS SYRITVRQL ESMIRLSEAI ARANCVDEIT PSFIAEAYDL LRQSIIRVDV DDVEMDEEFD NIESQSHAAS GNNDDNDDGT GSGVITSEPP ADIEEGQSE ATARPGTSEK KKTTVTYDKY VSMMNMIVRK IAEVDREGAE ELTAVDIVDW YLLQKENDLG SLAEYWEERR L AFKVIKRL VKDRILMEIH GTRHNLRDLE NEENENNKTV YVIHPNCEVL DQLEPQDSS

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Macromolecule #6: DNA replication licensing factor MCM7

MacromoleculeName: DNA replication licensing factor MCM7 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 95.049875 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MSAALPSIQL PVDYNNLFNE ITDFLVTFKQ DTLSSDATRN ENEDENLDAE NIEQHLLEKG PKYMAMLQKV ANRELNSVII DLDDILQYQ NEKFLQGTQA DDLVSAIQQN ANHFTELFCR AIDNNMPLPT KEIDYKDDVL DVILNQRRLR NERMLSDRTN E IRSENLMD ...String:
MSAALPSIQL PVDYNNLFNE ITDFLVTFKQ DTLSSDATRN ENEDENLDAE NIEQHLLEKG PKYMAMLQKV ANRELNSVII DLDDILQYQ NEKFLQGTQA DDLVSAIQQN ANHFTELFCR AIDNNMPLPT KEIDYKDDVL DVILNQRRLR NERMLSDRTN E IRSENLMD TTMDPPSSMN DALREVVEDE TELFPPNLTR RYFLYFKPLS QNCARRYRKK AISSKPLSVR QIKGDFLGQL IT VRGIITR VSDVKPAVEV IAYTCDQCGY EVFQEVNSRT FTPLSECTSE ECSQNQTKGQ LFMSTRASKF SAFQECKIQE LSQ QVPVGH IPRSLNIHVN GTLVRSLSPG DIVDVTGIFL PAPYTGFKAL KAGLLTETYL EAQFVRQHKK KFASFSLTSD VEER VMELI TSGDVYNRLA KSIAPEIYGN LDVKKALLLL LVGGVDKRVG DGMKIRGDIN VCLMGDPGVA KSQLLKAICK ISPRG VYTT GKGSSGVGLT AAVMKDPVTD EMILEGGALV LADNGICCID EFDKMDESDR TAIHEVMEQQ TISISKAGIN TTLNAR TSI LAAANPLYGR YNPRLSPLDN INLPAALLSR FDILFLMLDI PSRDDDEKLA EHVTYVHMHN KQPDLDFTPV EPSKMRE YI AYAKTKRPVM SEAVNDYVVQ AYIRLRQDSK REMDSKFSFG QATPRTLLGI IRLSQALAKL RLADMVDIDD VEEALRLV R VSKESLYQET NKSKEDESPT TKIFTIIKKM LQETGKNTLS YENIVKTVRL RGFTMLQLSN CIQEYSYLNV WHLINEGNT LKFVDDGTMD TDQEDSLVST PKLAPQTTAS ANVSAQDSDI DLQDA

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Macromolecule #7: Cell division control protein 7

MacromoleculeName: Cell division control protein 7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 58.391129 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: MTSKTKNIDD IPPEIKEEMI QLYHDLPGIE NEYKLIDKIG EGTFSSVYKA KDITGKITKK FASHFWNYGS NYVALKKIYV TSSPQRIYN ELNLLYIMTG SSRVAPLCDA KRVRDQVIAV LPYYPHEEFR TFYRDLPIKG IKKYIWELLR ALKFVHSKGI I HRDIKPTN ...String:
MTSKTKNIDD IPPEIKEEMI QLYHDLPGIE NEYKLIDKIG EGTFSSVYKA KDITGKITKK FASHFWNYGS NYVALKKIYV TSSPQRIYN ELNLLYIMTG SSRVAPLCDA KRVRDQVIAV LPYYPHEEFR TFYRDLPIKG IKKYIWELLR ALKFVHSKGI I HRDIKPTN FLFNLELGRG VLVDFGLAEA QMDYKSMISS QNDYDNYANT NHDGGYSMRN HEQFCPCIMR NQYSPNSHNQ TP PMVTIQN GKVVHLNNVN GVDLTKGYPK NETRRIKRAN RAGTRGFRAP EVLMKCGAQS TKIDIWSVGV ILLSLLGRRF PMF QSLDDA DSLLELCTIF GWKELRKCAA LHGLGFEASG LIWDKPNGYS NGLKEFVYDL LNKECTIGTF PEYSVAFETF GFLQ QELHD RMSIEPQLPD PKTNMDAVDA YELKKYQEEI WSDHYWCFQV LEQCFEMDPQ KRSSAEDLLK TPFFNELNEN TYLLD GEST DEDDVVSSSE ADLLDKDVLL ISE

+
Macromolecule #8: DDK kinase regulatory subunit DBF4

MacromoleculeName: DDK kinase regulatory subunit DBF4 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 84.503305 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288C (yeast)
SequenceString: KRRWKKNFIA VSAANRFKKI SSSGALENLY FQGEMVSPTK MIIASPAKET DTNLKHNNGI AASTTAAGHL NVFSNDNNCN NNNTTESFP KKRSLEALEA QQQQHLHEKK RARIERARSI EGAVQVSKGT GLKNVEPRVT PKELLEWQTN WKKIMKRDSR I YFDITDDV ...String:
KRRWKKNFIA VSAANRFKKI SSSGALENLY FQGEMVSPTK MIIASPAKET DTNLKHNNGI AASTTAAGHL NVFSNDNNCN NNNTTESFP KKRSLEALEA QQQQHLHEKK RARIERARSI EGAVQVSKGT GLKNVEPRVT PKELLEWQTN WKKIMKRDSR I YFDITDDV EMNTYNKSKM DKRRDLLKRG FLTLGAQITQ FFDTTVTIVI TRRSVENIYL LKDTDILSRA KKNYMKVWSY EK AARFLKN LDVDLDHLSK TKSASLAAPT LSNLLHNEKL YGPTDRDPRT KRDDIHYFKY PHVYLYDLWQ TWAPIITLEW KPQ ELTNLD ELPYPILKIG SFGRCPFIGD RNYDESSYKR VVKRYSRDKA NKKYALQLRA LFQYHADTLL NTSSVNDQTK NLIF IPHTC NDSTKSFKKW MQEKAKNFEK TELKKTDDSA VQDVRNEHAD QTDEKNSILL NETETKEPPL KEEKENKQSI AEESN KYPQ RKELAATPKL NHPVLATFAR QETEEVPDDL CTLKTKSRQA FEIKASGAHQ SNDVATSFGN GLGPTRASVM SKNMKS LSR LMVDRKLGVK QTNGNNKNYT ATIATTAETS KENRHRLDFN ALKKDEAPSK ETGKDSAVHL ETNRKPQNFP KVATKSV SA DSKVHNDIKI TTTESPTASK KSTSTNVTLH FNAQTAQTAQ PVKKETVKNS GYCENCRVKY ESLEQHIVSE KHLSFAEN D LNFEAIDSLI ENLRFQI

+
Macromolecule #9: DNA (53-MER)

MacromoleculeName: DNA (53-MER) / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 16.326441 KDa
SequenceString: (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC)(DT) (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA) (DT)(DG)(DC)(DG)(DC)(DA) ...String:
(DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC)(DT) (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA) (DT)(DG)(DC)(DG)(DC)(DA)(DT)(DG) (DC)(DA)(DT)(DG)(DC)

+
Macromolecule #10: DNA (53-MER)

MacromoleculeName: DNA (53-MER) / type: dna / ID: 10 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 16.335457 KDa
SequenceString: (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA)(DT)(DG)(DC)(DA)(DG)(DC)(DA)(DT) (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA) (DT)(DG)(DC)(DG)(DC)(DA) ...String:
(DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA)(DT)(DG)(DC)(DA)(DG)(DC)(DA)(DT) (DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA) (DT)(DG)(DC)(DG)(DC)(DA)(DT)(DG) (DC)(DA)(DT)(DG)(DC)

+
Macromolecule #11: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 11 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

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

+
Macromolecule #12: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 12 / Number of copies: 10 / Formula: MG
Molecular weightTheoretical: 24.305 Da

+
Macromolecule #13: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 13 / Number of copies: 11 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

+
Macromolecule #14: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 14 / Number of copies: 8 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.6
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: blotted for 3 seconds before plunging.
DetailsThe entire MCM loading and phosphorylation reaction was applied to the EM grid.

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.1 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 18135 / Average exposure time: 9.0 sec. / Average electron dose: 51.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: NONE / Details: CryoSPARC ab initio model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 149876
FSC plot (resolution estimation)

-
Atomic model buiding 1

Initial model(PDB ID:

6f0l

,

5bk4

,

6eyc

,

6ya7

,

3qbz

)
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-7p5z:
Structure of a DNA-loaded MCM double hexamer engaged with the Dbf4-dependent kinase

+
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