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- EMDB-13624: Structure of MCM2-7 DH complexed with Cdc7-Dbf4 in the presence o... -

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Basic information

Entry
Database: EMDB / ID: EMD-13624
TitleStructure of MCM2-7 DH complexed with Cdc7-Dbf4 in the presence of ATPgS, state III (3D auto-refined map)
Map dataC1 3D auto-refinement of MD-(ATPgS) state III
Sample
  • Complex: MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4
    • 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: DNA replication licensing factor MCM5
    • 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
KeywordsHelicase / Activation / Kinase / Phosphorylation / REPLICATION
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 / mitotic DNA replication checkpoint signaling / replication fork protection complex / mitotic DNA replication initiation / single-stranded DNA helicase activity / regulation of DNA-templated DNA replication initiation / silent mating-type cassette heterochromatin formation / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / DNA replication origin binding / nuclear replication fork / 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 (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSaleh A / Noguchi Y / Aramayo R / Ivanova ME / Speck C
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T005378/1 United Kingdom
Wellcome Trust107903/Z/15/Z United Kingdom
Medical Research Council (MRC, United Kingdom)A652-5PY40 United Kingdom
Wellcome Trust206175/Z/17/Z United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer.
Authors: Almutasem Saleh / Yasunori Noguchi / Ricardo Aramayo / Marina E Ivanova / Kathryn M Stevens / Alex Montoya / S Sunidhi / Nicolas Lopez Carranza / Marcin J Skwark / Christian Speck /
Abstract: The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at ...The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.
History
DepositionSep 27, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13624.png

Downloads & links

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Map

FileDownload / File: emd_13624.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC1 3D auto-refinement of MD-(ATPgS) state III
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.013
Minimum - Maximum-0.022848625 - 0.07325887
Average (Standard dev.)0.0001782211 (±0.0030334576)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 381.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13624_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 1 of C1 3D auto-refinement of MD-(ATPgS) state III

Fileemd_13624_half_map_1.map
Annotationhalf-map 1 of C1 3D auto-refinement of MD-(ATPgS) state III
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 2 of C1 3D auto-refinement of MD-(ATPgS) state III

Fileemd_13624_half_map_2.map
Annotationhalf-map 2 of C1 3D auto-refinement of MD-(ATPgS) state III
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4

EntireName: MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4
Components
  • Complex: MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4
    • 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: DNA replication licensing factor MCM5
    • 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

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Supramolecule #1: MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4

SupramoleculeName: MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 1.5 MDa

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Macromolecule #1: DNA replication licensing factor MCM2

MacromoleculeName: DNA replication licensing factor MCM2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's 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

UniProtKB: DNA replication licensing factor MCM2

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Macromolecule #2: DNA replication licensing factor MCM3

MacromoleculeName: DNA replication licensing factor MCM3 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MEGSTGFDGD ATTFFAPDAV FGDRVRRFQE FLDTFTSYRD SVRSIQVYNS NNAANYNDDQ DDADERDLLG DDDGDDLEKE KKAASSTSL NILPHRIIIS LDDLREFDRS FWSGILVEPA YFIPPAEKAL TDLADSMDDV PHPNASAVSS RHPWKLSFKG S FGAHALSP ...String:
MEGSTGFDGD ATTFFAPDAV FGDRVRRFQE FLDTFTSYRD SVRSIQVYNS NNAANYNDDQ DDADERDLLG DDDGDDLEKE KKAASSTSL NILPHRIIIS LDDLREFDRS FWSGILVEPA YFIPPAEKAL TDLADSMDDV PHPNASAVSS RHPWKLSFKG S FGAHALSP RTLTAQHLNK LVSVEGIVTK TSLVRPKLIR SVHYAAKTGR FHYRDYTDAT TTLTTRIPTP AIYPTEDTEG NK LTTEYGY STFIDHQRIT VQEMPEMAPA GQLPRSIDVI LDDDLVDKTK PGDRVNVVGV FKSLGAGGMN QSNSNTLIGF KTL ILGNTV YPLHARSTGV AARQMLTDFD IRNINKLSKK KDIFDILSQS LAPSIYGHDH IKKAILLMLM GGVEKNLENG SHLR GDINI LMVGDPSTAK SQLLRFVLNT ASLAIATTGR GSSGVGLTAA VTTDRETGER RLEAGAMVLA DRGVVCIDEF DKMTD VDRV AIHEVMEQQT VTIAKAGIHT TLNARCSVIA AANPVFGQYD VNRDPHQNIA LPDSLLSRFD LLFVVTDDIN EIRDRS ISE HVLRTHRYLP PGYLEGEPVR ERLNLSLAVG EDADINPEEH SNSGAGVENE GEDDEDHVFE KFNPLLQAGA KLAKNKG NY NGTEIPKLVT IPFLRKYVQY AKERVIPQLT QEAINVIVKN YTDLRNDDNT KKSPITARTL ETLIRLATAH AKVRLSKT V NKVDAKVAAN LLRFALLGED IGNDIDEEES EYEEALSKRS PQKSPKKRQR VRQPASNSGS PIKSTPRRST ASSVNATPS SARRILRFQD DEQNAGEDDN DIMSPLPADE EAELQRRLQL GLRVSPRRRE HLHAPEEGSS GPLTEVGTPR LPNVSSAGQD DEQQQSVIS FDNVEPGTIS TGRLSLISGI IARLMQTEIF EEESYPVASL FERINEELPE EEKFSAQEYL AGLKIMSDRN N LMVADDKV WRV

UniProtKB: DNA replication licensing factor MCM3

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Macromolecule #3: DNA replication licensing factor MCM4

MacromoleculeName: DNA replication licensing factor MCM4 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's 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

UniProtKB: DNA replication licensing factor MCM4

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Macromolecule #4: DNA replication licensing factor MCM5

MacromoleculeName: DNA replication licensing factor MCM5 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's 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

UniProtKB: Minichromosome maintenance protein 5

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Macromolecule #5: DNA replication licensing factor MCM6

MacromoleculeName: DNA replication licensing factor MCM6 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's 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

UniProtKB: DNA replication licensing factor MCM6

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Macromolecule #6: DNA replication licensing factor MCM7

MacromoleculeName: DNA replication licensing factor MCM7 / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's 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

UniProtKB: DNA replication licensing factor MCM7

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Macromolecule #7: Cell division control protein 7

MacromoleculeName: Cell division control protein 7 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's 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

UniProtKB: Cell division control protein 7

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Macromolecule #8: DDK kinase regulatory subunit DBF4

MacromoleculeName: DDK kinase regulatory subunit DBF4 / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVSPTKMIIR SPLKETDTNL KHNNGIAAST TAAGHLNVFS NDNNCNNNNT TESFPKKRSL ERLELQQQQH LHEKKRARIE RARSIEGAV QVSKGTGLKN VEPRVTPKEL LEWQTNWKKI MKRDSRIYFD ITDDVEMNTY NKSKMDKRRD LLKRGFLTLG A QITQFFDT ...String:
MVSPTKMIIR SPLKETDTNL KHNNGIAAST TAAGHLNVFS NDNNCNNNNT TESFPKKRSL ERLELQQQQH LHEKKRARIE RARSIEGAV QVSKGTGLKN VEPRVTPKEL LEWQTNWKKI MKRDSRIYFD ITDDVEMNTY NKSKMDKRRD LLKRGFLTLG A QITQFFDT TVTIVITRRS VENIYLLKDT DILSRAKKNY MKVWSYEKAA RFLKNLDVDL DHLSKTKSAS LAAPTLSNLL HN EKLYGPT DRDPRTKRDD IHYFKYPHVY LYDLWQTWAP IITLEWKPQE LTNLDELPYP ILKIGSFGRC PFIGDRNYDE SSY KRVVKR YSRDKANKKY ALQLRALFQY HADTLLNTSS VNDQTKNLIF IPHTCNDSTK SFKKWMQEKA KNFEKTELKK TDDS AVQDV RNEHADQTDE KNSILLNETE TKEPPLKEEK ENKQSIAEES NKYPQRKELA ATPKLNHPVL ATFARQETEE VPDDL CTLK TKSRQAFEIK ASGAHQSNDV ATSFGNGLGP TRASVMSKNM KSLSRLMVDR KLGVKQTNGN NKNYTATIAT TAETSK ENR HRLDFNALKK DEAPSKETGK DSAVHLETNR KPQNFPKVAT KSVSADSKVH NDIKITTTES PTASKKSTST NVTLHFN AQ TAQTAQPVKK ETVKNSGYCE NCRVKYESLE QHIVSEKHLS FAENDLNFEA IDSLIENLRF QI

UniProtKB: DDK kinase regulatory subunit DBF4

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 1.5 seconds and blot force +2.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 9909 / Average electron dose: 45.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: An ab initio initial model was generated using RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 73093
FSC plot (resolution estimation)

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