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- EMDB-47473: Cryo-EM structure of human CMG helicase stalled at G4-containing ... -

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Basic information

Entry
Database: EMDB / ID: EMD-47473
TitleCryo-EM structure of human CMG helicase stalled at G4-containing DNA template
Map dataComposite Map
Sample
  • Complex: Human CMG, G4 stall state
    • Protein or peptide: x 11 types
    • DNA: x 2 types
  • Ligand: x 6 types
Keywordshelicase / DNA replication / cell division / fork stalling / translocation / REPLICATION / REPLICATION-DNA complex
Function / homology
Function and homology information


Switching of origins to a post-replicative state / Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / nuclear origin of replication recognition complex / alpha DNA polymerase:primase complex / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / mitotic DNA replication / DNA replication checkpoint signaling ...Switching of origins to a post-replicative state / Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / nuclear origin of replication recognition complex / alpha DNA polymerase:primase complex / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / mitotic DNA replication / DNA replication checkpoint signaling / regulation of phosphorylation / CMG complex / DNA replication preinitiation complex / MCM complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / regulation of DNA-templated DNA replication initiation / inner cell mass cell proliferation / DNA strand elongation involved in DNA replication / G1/S-Specific Transcription / DNA replication origin binding / cochlea development / DNA replication initiation / Activation of the pre-replicative complex / Activation of ATR in response to replication stress / cellular response to interleukin-4 / DNA helicase activity / cellular response to epidermal growth factor stimulus / Assembly of the pre-replicative complex / helicase activity / DNA-templated DNA replication / multicellular organism growth / cellular response to xenobiotic stimulus / Orc1 removal from chromatin / cellular senescence / nucleosome assembly / single-stranded DNA binding / histone binding / DNA helicase / chromosome, telomeric region / DNA replication / cell population proliferation / cilium / ciliary basal body / intracellular membrane-bounded organelle / apoptotic process / DNA damage response / centrosome / chromatin binding / chromatin / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / identical protein binding / nucleus / membrane / cytoplasm / cytosol
Similarity search - Function
DNA replication licensing factor MCM2-like, winged-helix domain / : / PSF2 N-terminal domain / : / : / PSF3 N-terminal domain / PSF1 C-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 ...DNA replication licensing factor MCM2-like, winged-helix domain / : / PSF2 N-terminal domain / : / : / PSF3 N-terminal domain / PSF1 C-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / GINS complex, subunit Psf3 superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / GINS complex, subunit Psf1 / GINS, helical bundle-like domain superfamily / : / MCM5, C-terminal domain / DNA replication licensing factor MCM7, winged helix / DNA replication licensing factor Mcm5 / MCM4, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / : / MCM3-like, winged helix domain / 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 / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Cell division control protein 45 homolog / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM6 / DNA replication complex GINS protein PSF1 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae W303 (yeast) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsAllwein B / Batra S / Remus D / Hite R
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM152094 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM126907 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30 CA008748 United States
CitationJournal: Science / Year: 2025
Title: G-quadruplex-stalled eukaryotic replisome structure reveals helical inchworm DNA translocation.
Authors: Sahil Batra / Benjamin Allwein / Charanya Kumar / Sujan Devbhandari / Jan-Gert Brüning / Soon Bahng / Chong M Lee / Kenneth J Marians / Richard K Hite / Dirk Remus /
Abstract: DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we characterized fork ...DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we characterized fork collisions with preformed G4s in the parental DNA using reconstituted yeast and human replisomes. We demonstrate that a single G4 in the leading strand template is sufficient to stall replisomes by arresting the CMG helicase. Cryo-electron microscopy structures of stalled yeast and human CMG complexes reveal that the folded G4 is lodged inside the central CMG channel, arresting translocation. The G4 stabilizes the CMG at distinct translocation intermediates, suggesting an unprecedented helical inchworm mechanism for DNA translocation. These findings illuminate the eukaryotic replication fork mechanism under normal and perturbed conditions.
History
DepositionOct 23, 2024-
Header (metadata) releaseMar 26, 2025-
Map releaseMar 26, 2025-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47473.png

Downloads & links

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Map

FileDownload / File: emd_47473.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.83 Å/pix.
x 448 pix.
= 370.048 Å		0.83 Å/pix.
x 448 pix.
= 370.048 Å		0.83 Å/pix.
x 448 pix.
= 370.048 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0
Minimum - Maximum-39.674550000000004 - 100.950019999999995
Average (Standard dev.)0.0073895156 (±1.3309585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 370.04797 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47473_msk_1.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 CMG, G4 stall state

EntireName: Human CMG, G4 stall state
Components
  • Complex: Human CMG, G4 stall state
    • Protein or peptide: DNA replication complex GINS protein PSF1
    • Protein or peptide: DNA replication complex GINS protein PSF2
    • Protein or peptide: DNA replication complex GINS protein PSF3
    • Protein or peptide: DNA replication complex GINS protein SLD5
    • Protein or peptide: Cell division control protein 45 homolog
    • DNA: Leading strand DNA template
    • DNA: Lagging strand DNA template
    • Protein or peptide: Isoform 2 of DNA replication licensing factor MCM3
    • Protein or peptide: DNA replication licensing factor MCM6
    • Protein or peptide: DNA replication licensing factor MCM5
    • Protein or peptide: DNA replication licensing factor MCM2
    • Protein or peptide: DNA replication licensing factor MCM4
    • Protein or peptide: DNA replication licensing factor MCM7
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ZINC ION
  • Ligand: POTASSIUM ION
  • Ligand: water

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Supramolecule #1: Human CMG, G4 stall state

SupramoleculeName: Human CMG, G4 stall state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #7-#13, #2, #5, #4, #1, #3, #6
Details: Human CMG stalled at G4-containing DNA template, lacking the fork protection complex (TIMELESS-TIPIN)
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 586.01 KDa

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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: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 102.034102 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAESSESFTM ASSPAQRRRG NDPLTSSPGR SSRRTDALTS SPGRDLPPFE DESEGLLGTE GPLEEEEDGE ELIGDGMERD YRAIPELDA YEAEGLALDD EDVEELTASQ REAAERAMRQ RDREAGRGLG RMRRGLLYDS DEEDEERPAR KRRQVERATE D GEEDEEMI ...String:
MAESSESFTM ASSPAQRRRG NDPLTSSPGR SSRRTDALTS SPGRDLPPFE DESEGLLGTE GPLEEEEDGE ELIGDGMERD YRAIPELDA YEAEGLALDD EDVEELTASQ REAAERAMRQ RDREAGRGLG RMRRGLLYDS DEEDEERPAR KRRQVERATE D GEEDEEMI ESIENLEDLK GHSVREWVSM AGPRLEIHHR FKNFLRTHVD SHGHNVFKER ISDMCKENRE SLVVNYEDLA AR EHVLAYF LPEAPAELLQ IFDEAALEVV LAMYPKYDRI TNHIHVRISH LPLVEELRSL RQLHLNQLIR TSGVVTSCTG VLP QLSMVK YNCNKCNFVL GPFCQSQNQE VKPGSCPECQ SAGPFEVNME ETIYQNYQRI RIQESPGKVA AGRLPRSKDA ILLA DLVDS CKPGDEIELT GIYHNNYDGS LNTANGFPVF ATVILANHVA KKDNKVAVGE LTDEDVKMIT SLSKDQQIGE KIFAS IAPS IYGHEDIKRG LALALFGGEP KNPGGKHKVR GDINVLLCGD PGTAKSQFLK YIEKVSSRAI FTTGQGASAV GLTAYV QRH PVSREWTLEA GALVLADRGV CLIDEFDKMN DQDRTSIHEA MEQQSISISK AGIVTSLQAR CTVIAAANPI GGRYDPS LT FSENVDLTEP IISRFDILCV VRDTVDPVQD EMLARFVVGS HVRHHPSNKE EEGLANGSAA EPAMPNTYGV EPLPQEVL K KYIIYAKERV HPKLNQMDQD KVAKMYSDLR KESMATGSIP ITVRHIESMI RMAEAHARIH LRDYVIEDDV NMAIRVMLE SFIDTQKFSV MRSMRKTFAR YLSFRRDNNE LLLFILKQLV AEQVTYQRNR FGAQQDTIEV PEKDLVDKAR QINIHNLSAF YDSELFRMN KFSHDLKRKM ILQQF

UniProtKB: DNA replication licensing factor MCM2

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Macromolecule #2: Isoform 2 of DNA replication licensing factor MCM3

MacromoleculeName: Isoform 2 of DNA replication licensing factor MCM3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 96.04332 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MLPRSPPLPR GNLWWREEFG SFRAGVESSW EPPRDFGGGS SLAAGMAGTV VLDDVELREA QRDYLDFLDD EEDQGIYQSK VRELISDNQ YRLIVNVNDL RRKNEKRANR LLNNAFEELV AFQRALKDFV ASIDATYAKQ YEEFYVGLEG SFGSKHVSPR T LTSCFLSC ...String:
MLPRSPPLPR GNLWWREEFG SFRAGVESSW EPPRDFGGGS SLAAGMAGTV VLDDVELREA QRDYLDFLDD EEDQGIYQSK VRELISDNQ YRLIVNVNDL RRKNEKRANR LLNNAFEELV AFQRALKDFV ASIDATYAKQ YEEFYVGLEG SFGSKHVSPR T LTSCFLSC VVCVEGIVTK CSLVRPKVVR SVHYCPATKK TIERRYSDLT TLVAFPSSSV YPTKDEENNP LETEYGLSVY KD HQTITIQ EMPEKAPAGQ LPRSVDVILD DDLVDKAKPG DRVQVVGTYR CLPGKKGGYT SGTFRTVLIA CNVKQMSKDA QPS FSAEDI AKIKKFSKTR SKDIFDQLAK SLAPSIHGHD YVKKAILCLL LGGVERDLEN GSHIRGDINI LLIGDPSVAK SQLL RYVLC TAPRAIPTTG RGSSGVGLTA AVTTDQETGE RRLEAGAMVL ADRGVVCIDE FDKMSDMDRT AIHEVMEQGR VTIAK AGIH ARLNARCSVL AAANPVYGRY DQYKTPMENI GLQDSLLSRF DLLFIMLDQM DPEQDREISD HVLRMHRYRA PGEQDG DAM PLGSAVDILA TDDPNFSQED QQDTQIYEKH DNLLHGTKKK KEKMVSAAFM KKYIHVAKII KPVLTQESAT YIAEEYS RL RSQDSMSSDT ARTSPVTART LETLIRLATA HAKARMSKTV DLQDAEEAVE LVQYAYFKKV LEKEKKRKKR SEDESETE D EEEKSQEDQE QKRKRRKTRQ PDAKDGDSYD PYDFSDTEEE MPQVHTPKTA DSQETKESQK VELSESRLKA FKVALLDVF REAHAQSIGM NRLTESINRD SEEPFSSVEI QAALSKMQDD NQVMVSEGII FLI

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 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.119461 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MHHHHHHHHE NLYFQGSSAT MSSPASTPSR RGSRRGRATP AQTPRSEDAR SSPSQRRRGE DSTSTGELQP MPTSPGVDLQ SPAAQDVLF SSPPQMHSSA IPLDFDVSSP LTYGTPSSRV EGTPRSGVRG TPVRQRPDLG SAQKGLQVDL QSDGAAAEDI V ASEQSLGQ ...String:
MHHHHHHHHE NLYFQGSSAT MSSPASTPSR RGSRRGRATP AQTPRSEDAR SSPSQRRRGE DSTSTGELQP MPTSPGVDLQ SPAAQDVLF SSPPQMHSSA IPLDFDVSSP LTYGTPSSRV EGTPRSGVRG TPVRQRPDLG SAQKGLQVDL QSDGAAAEDI V ASEQSLGQ KLVIWGTDVN VAACKENFQR FLQRFIDPLA KEEENVGIDI TEPLYMQRLG EINVIGEPFL NVNCEHIKSF DK NLYRQLI SYPQEVIPTF DMAVNEIFFD RYPDSILEHQ IQVRPFNALK TKNMRNLNPE DIDQLITISG MVIRTSQLIP EMQ EAFFQC QVCAHTTRVE MDRGRIAEPS VCGRCHTTHS MALIHNRSLF SDKQMIKLQE SPEDMPAGQT PHTVILFAHN DLVD KVQPG DRVNVTGIYR AVPIRVNPRV SNVKSVYKTH IDVIHYRKTD AKRLHGLDEE AEQKLFSEKR VELLKELSRK PDIYE RLAS ALAPSIYEHE DIKKGILLQL FGGTRKDFSH TGRGKFRAEI NILLCGDPGT SKSQLLQYVY NLVPRGQYTS GKGSSA VGL TAYVMKDPET RQLVLQTGAL VLSDNGICCI DEFDKMNEST RSVLHEVMEQ QTLSIAKAGI ICQLNARTSV LAAANPI ES QWNPKKTTIE NIQLPHTLLS RFDLIFLLLD PQDEAYDRRL AHHLVALYYQ SEEQAEEELL DMAVLKDYIA YAHSTIMP R LSEEASQALI EAYVDMRKIG SSRGMVSAYP RQLESLIRLA EAHAKVRLSN KVEAIDVEEA KRLHREALKQ SATDPRTGI VDISILTTGM SATSRKRKEE LAEALKKLIL SKGKTPALKY QQLFEDIRGQ SDIAITKDMF EEALRALADD DFLTVTGKTV RLL

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 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 82.406633 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSGFDDPGIF YSDSFGGDAQ ADEGQARKSQ LQRRFKEFLR QYRVGTDRTG FTFKYRDELK RHYNLGEYWI EVEMEDLASF DEDLADYLY KQPAEHLQLL EEAAKEVADE VTRPRPSGEE VLQDIQVMLK SDASPSSIRS LKSDMMSHLV KIPGIIIAAS A VRAKATRI ...String:
MSGFDDPGIF YSDSFGGDAQ ADEGQARKSQ LQRRFKEFLR QYRVGTDRTG FTFKYRDELK RHYNLGEYWI EVEMEDLASF DEDLADYLY KQPAEHLQLL EEAAKEVADE VTRPRPSGEE VLQDIQVMLK SDASPSSIRS LKSDMMSHLV KIPGIIIAAS A VRAKATRI SIQCRSCRNT LTNIAMRPGL EGYALPRKCN TDQAGRPKCP LDPYFIMPDK CKCVDFQTLK LQELPDAVPH GE MPRHMQL YCDRYLCDKV VPGNRVTIMG IYSIKKFGLT TSRGRDRVGV GIRSSYIRVL GIQVDTDGSG RSFAGAVSPQ EEE EFRRLA ALPNVYEVIS KSIAPSIFGG TDMKKAIACL LFGGSRKRLP DGLTRRGDIN LLMLGDPGTA KSQLLKFVEK CSPI GVYTS GKGSSAAGLT ASVMRDPSSR NFIMEGGAMV LADGGVVCID EFDKMREDDR VAIHEAMEQQ TISIAKAGIT TTLNS RCSV LAAANSVFGR WDETKGEDNI DFMPTILSRF DMIFIVKDEH NEERDVMLAK HVITLHVSAL TQTQAVEGEI DLAKLK KFI AYCRVKCGPR LSAEAAEKLK NRYIIMRSGA RQHERDSDRR SSIPITVRQL EAIVRIAEAL SKMKLQPFAT EADVEEA LR LFQVSTLDAA LSGTLSGVEG FTSQEDQEML SRIEKQLKRR FAIGSQVSEH SIIKDFTKQK YPEHAIHKVL QLMLRRGE I QHRMQRKVLY RLK

UniProtKB: DNA replication licensing factor MCM5

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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: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 93.010273 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDLAAAAEPG AGSQHLEVRD EVAEKCQKLF LDFLEEFQSS DGEIKYLQLA EELIRPERNT LVVSFVDLEQ FNQQLSTTIQ EEFYRVYPY LCRALKTFVK DRKEIPLAKD FYVAFQDLPT RHKIRELTSS RIGLLTRISG QVVRTHPVHP ELVSGTFLCL D CQTVIRDV ...String:
MDLAAAAEPG AGSQHLEVRD EVAEKCQKLF LDFLEEFQSS DGEIKYLQLA EELIRPERNT LVVSFVDLEQ FNQQLSTTIQ EEFYRVYPY LCRALKTFVK DRKEIPLAKD FYVAFQDLPT RHKIRELTSS RIGLLTRISG QVVRTHPVHP ELVSGTFLCL D CQTVIRDV EQQFKYTQPN ICRNPVCANR RRFLLDTNKS RFVDFQKVRI QETQAELPRG SIPRSLEVIL RAEAVESAQA GD KCDFTGT LIVVPDVSKL STPGARAETN SRVSGVDGYE TEGIRGLRAL GVRDLSYRLV FLACCVAPTN PRFGGKELRD EEQ TAESIK NQMTVKEWEK VFEMSQDKNL YHNLCTSLFP TIHGNDEVKR GVLLMLFGGV PKTTGEGTSL RGDINVCIVG DPST AKSQF LKHVEEFSPR AVYTSGKASS AAGLTAAVVR DEESHEFVIE AGALMLADNG VCCIDEFDKM DVRDQVAIHE AMEQQ TISI TKAGVKATLN ARTSILAAAN PISGHYDRSK SLKQNINLSA PIMSRFDLFF ILVDECNEVT DYAIARRIVD LHSRIE ESI DRVYSLDDIR RYLLFARQFK PKISKESEDF IVEQYKHLRQ RDGSGVTKSS WRITVRQLES MIRLSEAMAR MHCCDEV QP KHVKEAFRLL NKSIIRVETP DVNLDQEEEI QMEVDEGAGG INGHADSPAP VNGINGYNED INQESAPKAS LRLGFSEY C RISNLIVLHL RKVEEEEDES ALKRSELVNW YLKEIESEID SEEELINKKR IIEKVIHRLT HYDHVLIELT QAGLKGSTE GSESYEEDPY LVVNPNYLLE D

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 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 81.411875 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MALKDYALEK EKVKKFLQEF YQDDELGKKQ FKYGNQLVRL AHREQVALYV DLDDVAEDDP ELVDSICENA RRYAKLFADA VQELLPQYK EREVVNKDVL DVYIEHRLMM EQRSRDPGMV RSPQNQYPAE LMRRFELYFQ GPSSNKPRVI REVRADSVGK L VTVRGIVT ...String:
MALKDYALEK EKVKKFLQEF YQDDELGKKQ FKYGNQLVRL AHREQVALYV DLDDVAEDDP ELVDSICENA RRYAKLFADA VQELLPQYK EREVVNKDVL DVYIEHRLMM EQRSRDPGMV RSPQNQYPAE LMRRFELYFQ GPSSNKPRVI REVRADSVGK L VTVRGIVT RVSEVKPKMV VATYTCDQCG AETYQPIQSP TFMPLIMCPS QECQTNRSGG RLYLQTRGSR FIKFQEMKMQ EH SDQVPVG NIPRSITVLV EGENTRIAQP GDHVSVTGIF LPILRTGFRQ VVQGLLSETY LEAHRIVKMN KSEDDESGAG ELT REELRQ IAEEDFYEKL AASIAPEIYG HEDVKKALLL LLVGGVDQSP RGMKIRGNIN ICLMGDPGVA KSQLLSYIDR LAPR SQYTT GRGSSGVGLT AAVLRDSVSG ELTLEGGALV LADQGVCCID EFDKMAEADR TAIHEVMEQQ TISIAKAGIL TTLNA RCSI LAAANPAYGR YNPRRSLEQN IQLPAALLSR FDLLWLIQDR PDRDNDLRLA QHITYVHQHS RQPPSQFEPL DMKLMR RYI AMCREKQPMV PESLADYITA AYVEMRREAW ASKDATYTSA RTLLAILRLS TALARLRMVD VVEKEDVNEA IRLMEMS KD SLLGDKGQTA RTQRPADVIF ATVRELVSGG RSVRFSEAEQ RCVSRGFTPA QFQAALDEYE ELNVWQVNAS RTRITFV

UniProtKB: DNA replication licensing factor MCM7

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Macromolecule #7: DNA replication complex GINS protein PSF1

MacromoleculeName: DNA replication complex GINS protein PSF1 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.022469 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MFCEKAMELI RELHRAPEGQ LPAFNEDGLR QVLEEMKALY EQNQSDVNEA KSGGRSDLIP TIKFRHCSLL RNRRCTVAYL YDRLLRIRA LRWEYGSVLP NALRFHMAAE EMEWFNNYKR SLATYMRSLG GDEGLDITQD MKPPKSLYIE VRCLKDYGEF E VDDGTSVL ...String:
MFCEKAMELI RELHRAPEGQ LPAFNEDGLR QVLEEMKALY EQNQSDVNEA KSGGRSDLIP TIKFRHCSLL RNRRCTVAYL YDRLLRIRA LRWEYGSVLP NALRFHMAAE EMEWFNNYKR SLATYMRSLG GDEGLDITQD MKPPKSLYIE VRCLKDYGEF E VDDGTSVL LKKNSQHFLP RWKCEQLIRQ GVLEHILS

UniProtKB: DNA replication complex GINS protein PSF1

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Macromolecule #8: DNA replication complex GINS protein PSF2

MacromoleculeName: DNA replication complex GINS protein PSF2 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.336793 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDAAEVEFLA EKELVTIIPN FSLDKIYLIG GDLGPFNPGL PVEVPLWLAI NLKQRQKCRL LPPEWMDVEK LEKMRDHERK EETFTPMPS PYYMELTKLL LNHASDNIPK ADEIRTLVKD MWDTRIAKLR VSADSFVRQQ EAHAKLDNLT LMEINTSGTF L TQALNHMY ...String:
MDAAEVEFLA EKELVTIIPN FSLDKIYLIG GDLGPFNPGL PVEVPLWLAI NLKQRQKCRL LPPEWMDVEK LEKMRDHERK EETFTPMPS PYYMELTKLL LNHASDNIPK ADEIRTLVKD MWDTRIAKLR VSADSFVRQQ EAHAKLDNLT LMEINTSGTF L TQALNHMY KLRTNLQPLE STQSQDFENL YFQGSAWSHP QFEKGGGSGG GSGGSAWSHP QFEK

UniProtKB: DNA replication complex GINS protein PSF2

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Macromolecule #9: DNA replication complex GINS protein PSF3

MacromoleculeName: DNA replication complex GINS protein PSF3 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.562611 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSEAYFRVES GALGPEENFL SLDDILMSHE KLPVRTETAM PRLGAFFLER SAGAETDNAV PQGSKLELPL WLAKGLFDNK RRILSVELP KIYQEGWRTV FSADPNVVDL HKMGPHFYGF GSQLLHFDSP ENADISQSLL QTFIGRFRRI MDSSQNAYNE D TSALVARL ...String:
MSEAYFRVES GALGPEENFL SLDDILMSHE KLPVRTETAM PRLGAFFLER SAGAETDNAV PQGSKLELPL WLAKGLFDNK RRILSVELP KIYQEGWRTV FSADPNVVDL HKMGPHFYGF GSQLLHFDSP ENADISQSLL QTFIGRFRRI MDSSQNAYNE D TSALVARL DEMERGLFQT GQKGLNDFQC WEKGQASQIT ASNLVQNYKK RKFTDMED

UniProtKB: DNA replication complex GINS protein PSF3

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Macromolecule #10: DNA replication complex GINS protein SLD5

MacromoleculeName: DNA replication complex GINS protein SLD5 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.081873 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MTEEVDFLGQ DSDGGSEEVV LTPAELIERL EQAWMNEKFA PELLESKPEI VECVMEQLEH MEENLRRAKR EDLKVSIHQM EMERIRYVL SSYLRCRLMK IEKFFPHVLE KEKTRPEGEP SSLSPEELAF AREFMANTES YLKNVALKHM PPNLQKVDLF R AVPKPDLD ...String:
MTEEVDFLGQ DSDGGSEEVV LTPAELIERL EQAWMNEKFA PELLESKPEI VECVMEQLEH MEENLRRAKR EDLKVSIHQM EMERIRYVL SSYLRCRLMK IEKFFPHVLE KEKTRPEGEP SSLSPEELAF AREFMANTES YLKNVALKHM PPNLQKVDLF R AVPKPDLD SYVFLRVRER QENILVEPDT DEQRDYVIDL EKGSQHLIRY KTIAPLVASG AVQLI

UniProtKB: DNA replication complex GINS protein SLD5

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Macromolecule #11: Cell division control protein 45 homolog

MacromoleculeName: Cell division control protein 45 homolog / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.651539 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MFVSDFRKEF YEVVQSQRVL LFVASDVDAL CACKILQALF QCDHVQYTLV PVSGWQELET AFLEHKEQFH YFILINCGAN VDLLDILQP DEDTIFFVCD THRPVNVVNV YNDTQIKLLI KQDDDLEVPA YEDIFRDEEE DEEHSGNDSD GSEPSEKRTR L EEEIVEQT ...String:
MFVSDFRKEF YEVVQSQRVL LFVASDVDAL CACKILQALF QCDHVQYTLV PVSGWQELET AFLEHKEQFH YFILINCGAN VDLLDILQP DEDTIFFVCD THRPVNVVNV YNDTQIKLLI KQDDDLEVPA YEDIFRDEEE DEEHSGNDSD GSEPSEKRTR L EEEIVEQT MRRRQRREWE ARRRDILFDY EQYEYHGTSS AMVMFELAWM LSKDLNDMLW WAIVGLTDQW VQDKITQMKY VT DVGVLQR HVSRHNHRNE DEENTLSVDC TRISFEYDLR LVLYQHWSLH DSLCNTSYTA ARFKLWSVHG QKRLQEFLAD MGL PLKQVK QKFQAMDISL KENLREMIEE SANKFGMKDM RVQTFSIHFG FKHKFLASDV VFATMSLMES PEKDGSGTDH FIQA LDSLS RSNLDKLYHG LELAKKQLRA TQQTIASCLC TNLVISQGPF LYCSLMEGTP DVMLFSRPAS LSLLSKHLLK SFVCS TKNR RCKLLPLVMA APLSMEHGTV TVVGIPPETD SSDRKNFFGR AFEKAAESTS SRMLHNHFDL SVIELKAEDR SKFLDA LIS LLS

UniProtKB: Cell division control protein 45 homolog

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Macromolecule #12: Leading strand DNA template

MacromoleculeName: Leading strand DNA template / type: dna / ID: 12 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.582038 KDa
SequenceString:
(DG)(DT)(DG)(DA)(DT)(DA)(DT)(DC)(DT)(DG) (DC)(DT)(DT)(DT)(DG)(DG)(DG)(DT)(DG)(DG) (DG)(DT)(DG)(DG)(DG)(DT)(DG)(DG)(DG) (DT)(DT)(DG)(DA)(DG)(DG)(DC)(DA)(DA)(DT) (DA)

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Macromolecule #13: Lagging strand DNA template

MacromoleculeName: Lagging strand DNA template / type: dna / ID: 13 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.968626 KDa
SequenceString:
(DA)(DA)(DG)(DC)(DA)(DG)(DA)(DT)(DA)(DT) (DC)(DA)(DC)

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Macromolecule #14: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 14 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Macromolecule #15: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 15 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #16: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 16 / Number of copies: 5 / 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 #17: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 17 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #18: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 18 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #19: water

MacromoleculeName: water / type: ligand / ID: 19 / Number of copies: 39 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration.01 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMC8H17KN2O4SHEPES-KOH
100.0 mMCH3CO2KPotassium acetate
10.0 mMMg(CH3COO)2Magnesium acetate
2.5 mMC4H10O2S2DTT
0.02 %H(C2H4O)nO(C6H4)C9H19NP-40S
100.0 uMC10H17N6O12P3AMP-PNP
5.0 mMC10H16N5O13P3ATP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.5
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 30 second wait time after sample application; 30 second blot time, blot force 0.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3325 / Average exposure time: 3.0 sec. / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 29000
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: 1587336
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.0) / Number images used: 187345
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.0.0)
Details: Stochastic gradient descent (cryoSPARC ab initio model generation)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.0.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: in silico model / Details: ModelAngelo
DetailsInitial fitting was performed de novo by ModelAngelo, then iteratively improved with ChimeraX/ISOLDE, Coot, and Phenix real-space refinement algorithms.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 49.99
Output model

PDB-9e2z:
Cryo-EM structure of human CMG helicase stalled at G4-containing DNA template

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