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

Basic information

Entry

Database: EMDB / ID: EMD-47473

• Title: Cryo-EM structure of human CMG helicase stalled at G4-containing DNA template
• Map data: Composite Map

Sample

• Complex: Human CMG, G4 stall state
  • Protein or peptide: x 11 types
  • DNA: x 2 types
• Ligand: x 6 types

• Keywords: helicase / DNA replication / cell division / fork stalling / translocation / REPLICATION / REPLICATION-DNA complex

Function / homology

Function:

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 / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / alpha DNA polymerase:primase complex / mitotic DNA replication / DNA replication checkpoint signaling / CMG complex / regulation of phosphorylation / 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 / cochlea development / G1/S-Specific Transcription / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / Activation of ATR in response to replication stress / cellular response to epidermal growth factor stimulus / cellular response to interleukin-4 / Assembly of the pre-replicative complex / multicellular organism growth / DNA-templated DNA replication / Orc1 removal from chromatin / cellular senescence / cellular response to xenobiotic stimulus / nucleosome assembly / single-stranded DNA binding / DNA helicase / histone binding / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / chromosome, telomeric region / cell population proliferation / DNA replication / ciliary basal body / cilium / intracellular membrane-bounded organelle / apoptotic process / centrosome / DNA damage response / 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 / cytosol / cytoplasm

Domain/homology:

DNA replication licensing factor MCM2-like, winged-helix domain / : / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / 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 / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / MCM3-like, winged helix domain / 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 / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase

Component:

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

• Biological species: Saccharomyces cerevisiae W303 (yeast) / Homo sapiens (human) / synthetic construct (others)
• Method: single particle reconstruction / cryo EM / Resolution: 2.6 Å
• Authors: Allwein B / Batra S / Remus D / Hite R

Funding support

United States, 3 items

Organization	Grant number	Country
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

• Citation: Journal: 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 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

Deposition	Oct 23, 2024	-
Header (metadata) release	Mar 26, 2025	-
Map release	Mar 26, 2025	-
Update	Mar 26, 2025	-
Current status	Mar 26, 2025	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_47473.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Composite Map
• Voxel size: X=Y=Z: 0.826 Å

Density

Contour Level	By AUTHOR: 3.0
Minimum - Maximum	-39.674550000000004 - 100.950019999999995
Average (Standard dev.)	0.0073895156 (±1.3309585)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Z Y X Origin 0 0 0 Dimensions 448 448 448 Spacing 448 448 448
Cell	A=B=C: 370.04797 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_47473_msk_1.map

Sample components

Entire : Human CMG, G4 stall state

• Entire: Name: 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

Supramolecule #1: Human CMG, G4 stall state

• Supramolecule: Name: 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 weight: Theoretical: 586.01 KDa

Macromolecule #1: DNA replication licensing factor MCM2

• Macromolecule: Name: 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 weight: Theoretical: 102.034102 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

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

• Macromolecule: Name: 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 weight: Theoretical: 96.04332 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #3: DNA replication licensing factor MCM4

• Macromolecule: Name: 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 weight: Theoretical: 99.119461 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #4: DNA replication licensing factor MCM5

• Macromolecule: Name: 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 weight: Theoretical: 82.406633 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #5: DNA replication licensing factor MCM6

• Macromolecule: Name: 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 weight: Theoretical: 93.010273 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #6: DNA replication licensing factor MCM7

• Macromolecule: Name: 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 weight: Theoretical: 81.411875 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #7: DNA replication complex GINS protein PSF1

• Macromolecule: Name: 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 weight: Theoretical: 23.022469 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #8: DNA replication complex GINS protein PSF2

• Macromolecule: Name: 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 weight: Theoretical: 25.336793 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #9: DNA replication complex GINS protein PSF3

• Macromolecule: Name: 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 weight: Theoretical: 24.562611 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #10: DNA replication complex GINS protein SLD5

• Macromolecule: Name: 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 weight: Theoretical: 26.081873 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #11: Cell division control protein 45 homolog

• Macromolecule: Name: 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 weight: Theoretical: 65.651539 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

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

Macromolecule #12: Leading strand DNA template

• Macromolecule: Name: Leading strand DNA template / type: dna / ID: 12 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: synthetic construct (others)
• Molecular weight: Theoretical: 12.582038 KDa

Sequence

String:

(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)

Macromolecule #13: Lagging strand DNA template

• Macromolecule: Name: Lagging strand DNA template / type: dna / ID: 13 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: synthetic construct (others)
• Molecular weight: Theoretical: 3.968626 KDa

Sequence

String:

(DA)(DA)(DG)(DC)(DA)(DG)(DA)(DT)(DA)(DT) (DC)(DA)(DC)

Macromolecule #14: ADENOSINE-5'-DIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 14 / Number of copies: 1 / Formula: ADP
• Molecular weight: Theoretical: 427.201 Da

Chemical component information

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

Macromolecule #15: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 15 / Number of copies: 6 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Macromolecule #16: ADENOSINE-5'-TRIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 16 / Number of copies: 5 / Formula: ATP
• Molecular weight: Theoretical: 507.181 Da

Chemical component information

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

Macromolecule #17: ZINC ION

• Macromolecule: Name: ZINC ION / type: ligand / ID: 17 / Number of copies: 4 / Formula: ZN
• Molecular weight: Theoretical: 65.409 Da

Macromolecule #18: POTASSIUM ION

• Macromolecule: Name: POTASSIUM ION / type: ligand / ID: 18 / Number of copies: 2 / Formula: K
• Molecular weight: Theoretical: 39.098 Da

Macromolecule #19: water

• Macromolecule: Name: water / type: ligand / ID: 19 / Number of copies: 39 / Formula: HOH
• Molecular weight: Theoretical: 18.015 Da

Chemical component information

ChemComp-HOH:
WATER

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: .01 mg/mL

Buffer

pH: 7.6
Component:

Concentration	Formula	Name
20.0 mM	C8H17KN2O4S	HEPES-KOH
100.0 mM	CH3CO2K	Potassium acetate
10.0 mM	Mg(CH3COO)2	Magnesium acetate
2.5 mM	C4H10O2S2	DTT
0.02 %	H(C2H4O)nO(C6H4)C9H19	NP-40S
100.0 uM	C10H17N6O12P3	AMP-PNP
5.0 mM	C10H16N5O13P3	ATP

• Grid: Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.5
• Vitrification: Cryogen 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.

Electron microscopy

• Microscope: TFS KRIOS
• Image recording: Film 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/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination 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 stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 1587336
• Startup model: Type of model: NONE
• Final reconstruction: Applied 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 assignment: Type: OTHER / Software - Name: cryoSPARC (ver. 4.0.0); Details: Stochastic gradient descent (cryoSPARC ab initio model generation)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.0)
• Final 3D classification: Software - Name: cryoSPARC (ver. 4.0.0)

Atomic model buiding 1

• Initial model: Chain - Source name: Other / Chain - Initial model type: in silico model / Details: ModelAngelo
• Details: Initial fitting was performed de novo by ModelAngelo, then iteratively improved with ChimeraX/ISOLDE, Coot, and Phenix real-space refinement algorithms.
• Refinement: Space: 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