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- EMDB-10230: Cryo-EM Structure of the Fork Protection Complex Bound to CMG at ... -

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Basic information

Entry
Database: EMDB / ID: EMD-10230
TitleCryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork - conformation 2 MCM CTD:ssDNA
Map dataConformation2_MCM-CTD-ssDNA
Sample
  • Complex: Conformation 2 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
    • Complex: CMG-Csm3-Tof1-Mrc1-Ctf4
      • Protein or peptide: DNA replication licensing factor MCM6
      • Protein or peptide: DNA replication licensing factor MCM7
      • Protein or peptide: DNA replication licensing factor MCM2
      • Protein or peptide: Minichromosome maintenance protein 5
      • Protein or peptide: DNA replication licensing factor MCM3
      • Protein or peptide: DNA replication licensing factor MCM4
    • Complex: DNA
      • DNA: ssDNA, leading-strand template
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Keywordsprotein-DNA complex / replisome / AAA+ helicase / CMG / GINS / fork DNA / MCM / fork protection complex / CIP-box / REPLICATION
Function / homology
Function and homology information


MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / 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 ...MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / 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 / single-stranded 3'-5' DNA helicase activity / nuclear pre-replicative complex / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / replication fork protection complex / mitotic DNA replication initiation / double-strand break repair via break-induced replication / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / single-stranded DNA helicase activity / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / nuclear replication fork / 3'-5' DNA helicase activity / DNA replication origin binding / subtelomeric heterochromatin formation / DNA replication initiation / heterochromatin formation / helicase activity / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
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 ...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 / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBaretic D / Jenkyn-Bedford M
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_1201/12 United Kingdom
CitationJournal: Mol Cell / Year: 2020
Title: Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork.
Authors: Domagoj Baretić / Michael Jenkyn-Bedford / Valentina Aria / Giuseppe Cannone / Mark Skehel / Joseph T P Yeeles /
Abstract: The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric ...The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 "grip" duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.
History
DepositionAug 16, 2019-
Header (metadata) releaseMay 6, 2020-
Map releaseMay 6, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sko
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10230.png

Downloads & links

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Map

FileDownload / File: emd_10230.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConformation2_MCM-CTD-ssDNA
Voxel sizeX=Y=Z: 1.049 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.026153427 - 0.07122159
Average (Standard dev.)-0.000115153394 (±0.0014348766)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 377.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0491.0491.049
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z377.640377.640377.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0260.071-0.000

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Supplemental data

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Sample components

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Entire : Conformation 2 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA

EntireName: Conformation 2 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
Components
  • Complex: Conformation 2 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
    • Complex: CMG-Csm3-Tof1-Mrc1-Ctf4
      • Protein or peptide: DNA replication licensing factor MCM6
      • Protein or peptide: DNA replication licensing factor MCM7
      • Protein or peptide: DNA replication licensing factor MCM2
      • Protein or peptide: Minichromosome maintenance protein 5
      • Protein or peptide: DNA replication licensing factor MCM3
      • Protein or peptide: DNA replication licensing factor MCM4
    • Complex: DNA
      • DNA: ssDNA, leading-strand template
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Conformation 2 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA

SupramoleculeName: Conformation 2 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7 / Details: Mcm2-7 C-tier domains (CTD) with ssDNA
Molecular weightTheoretical: 1.4 MDa

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Supramolecule #2: CMG-Csm3-Tof1-Mrc1-Ctf4

SupramoleculeName: CMG-Csm3-Tof1-Mrc1-Ctf4 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#6
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #7
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: DNA replication licensing factor MCM6

MacromoleculeName: DNA replication licensing factor MCM6 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 113.110211 KDa
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 #2: DNA replication licensing factor MCM7

MacromoleculeName: DNA replication licensing factor MCM7 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 95.049875 KDa
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 #3: DNA replication licensing factor MCM2

MacromoleculeName: DNA replication licensing factor MCM2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 98.911539 KDa
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 #4: Minichromosome maintenance protein 5

MacromoleculeName: Minichromosome maintenance protein 5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 86.505734 KDa
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 MCM3

MacromoleculeName: DNA replication licensing factor MCM3 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 107.653508 KDa
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 #6: DNA replication licensing factor MCM4

MacromoleculeName: DNA replication licensing factor MCM4 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 105.138375 KDa
Recombinant expressionOrganism: 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 #7: ssDNA, leading-strand template

MacromoleculeName: ssDNA, leading-strand template / type: dna / ID: 7
Details: Cy3-label at 5'-end. Five phosphorothioate backbone linkages between residues 80-85 at 3'-end. dT (residue 16) contains biotinylation site (iBiodT).
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 26.396836 KDa
SequenceString: (DT)(DA)(DG)(DA)(DG)(DT)(DA)(DG)(DG)(DA) (DA)(DG)(DT)(DG)(DA)(DT)(DG)(DG)(DT)(DA) (DA)(DG)(DT)(DG)(DA)(DT)(DT)(DA)(DG) (DA)(DG)(DA)(DA)(DT)(DT)(DG)(DG)(DA)(DG) (DA) (DG)(DT)(DG)(DT)(DG)(DT) ...String:
(DT)(DA)(DG)(DA)(DG)(DT)(DA)(DG)(DG)(DA) (DA)(DG)(DT)(DG)(DA)(DT)(DG)(DG)(DT)(DA) (DA)(DG)(DT)(DG)(DA)(DT)(DT)(DA)(DG) (DA)(DG)(DA)(DA)(DT)(DT)(DG)(DG)(DA)(DG) (DA) (DG)(DT)(DG)(DT)(DG)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)

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Macromolecule #8: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 8 / Number of copies: 5 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 5 / 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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaOAcsodium acetate
0.5 mMTCEPtris(2-carboxyethyl)phosphine
0.1 mMAMP-PNPadenylyl-imidodiphosphate
0.7 mMMg(OAc)2magnesium acetate
0.005 %TWEEN20Polyoxyethylene (20) sorbitan monolaurate
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 5 sec. / Pretreatment - Atmosphere: AIR / Details: plasma current 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: Three microlitres of sample was applied on a grid and incubated for 15-30 s at 4 degC before manually blotting with filter paper for 10 s and plunge-freezing in liquid ethane..
DetailsIn vitro reconstitution from individual components: CMG, Csm3-Tof1, Mrc1, Ctf4 and a DNA fork

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Number grids imaged: 2 / Number real images: 6682 / Average exposure time: 7.0 sec. / Average electron dose: 37.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 632000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5u8s

Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 182000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5u8s

chain_id: 2, source_name: PDB, initial_model_type: experimental model

5u8s

chain_id: 3, source_name: PDB, initial_model_type: experimental model

5u8s

chain_id: 4, source_name: PDB, initial_model_type: experimental model

5u8s

chain_id: 5, source_name: PDB, initial_model_type: experimental model

5u8s

chain_id: 6, source_name: PDB, initial_model_type: experimental model

5u8s

chain_id: 7, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 20 / Target criteria: FSC 0.5
Output model

PDB-6sko:
Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork - conformation 2 MCM CTD:ssDNA

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