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- PDB-6xty: CryoEM structure of human CMG bound to AND-1 (CMGA) -

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Entry
Database: PDB / ID: 6xty
TitleCryoEM structure of human CMG bound to AND-1 (CMGA)
Components
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 6
  • Cell division control protein 45 homolog
  • WD repeat and HMG-box DNA-binding protein 1
KeywordsREPLICATION / CMG / Helicase / ATPase / Replisome
Function / homology
Function and homology information


DNA replication checkpoint / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / alpha DNA polymerase:primase complex / regulation of chromatin silencing at telomere / mitotic DNA replication / nuclear origin of replication recognition complex / cellular response to xenobiotic stimulus / MCM complex ...DNA replication checkpoint / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / alpha DNA polymerase:primase complex / regulation of chromatin silencing at telomere / mitotic DNA replication / nuclear origin of replication recognition complex / cellular response to xenobiotic stimulus / MCM complex / negative regulation of DNA helicase activity / mitotic DNA replication initiation / inner cell mass cell proliferation / DNA replication preinitiation complex / nuclear pre-replicative complex / regulation of phosphorylation / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / double-strand break repair via break-induced replication / nuclear replication fork / replication fork protection complex / DNA strand elongation involved in DNA replication / DNA duplex unwinding / cochlea development / DNA unwinding involved in DNA replication / DNA replication origin binding / DNA replication initiation / positive regulation of G1/S transition of mitotic cell cycle / cellular response to interleukin-4 / regulation of transcription involved in G1/S transition of mitotic cell cycle / cellular response to epidermal growth factor stimulus / nucleosome assembly / microtubule cytoskeleton / chromatin / DNA helicase / DNA helicase activity / G1/S transition of mitotic cell cycle / DNA-dependent DNA replication / single-stranded DNA binding / cell population proliferation / mitotic cell cycle / nuclear chromosome, telomeric region / histone binding / DNA replication / response to drug / centrosome / DNA repair / apoptotic process / cellular response to DNA damage stimulus / chromatin binding / perinuclear region of cytoplasm / enzyme binding / DNA binding / membrane / nucleoplasm / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
DNA replication licensing factor Mcm3 / GINS complex subunit Sld5 / GINS subunit, domain A / WD40 repeat, conserved site / Mini-chromosome maintenance, conserved site / WD40-repeat-containing domain / Mini-chromosome maintenance complex protein 4 / Nucleic acid-binding, OB-fold / GINS complex, subunit Psf3 / High mobility group box domain ...DNA replication licensing factor Mcm3 / GINS complex subunit Sld5 / GINS subunit, domain A / WD40 repeat, conserved site / Mini-chromosome maintenance, conserved site / WD40-repeat-containing domain / Mini-chromosome maintenance complex protein 4 / Nucleic acid-binding, OB-fold / GINS complex, subunit Psf3 / High mobility group box domain / DNA replication licensing factor Mcm7 / Anaphase-promoting complex subunit 4, WD40 domain / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm5 / MCM domain / WD40 repeat / AAA+ ATPase domain / CDC45 family / GINS complex, subunit Psf1 / DNA replication complex GINS protein Psf2 / DNA replication licensing factor Mcm2 / Minichromosome loss protein Mcl1, middle region / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / WD40-repeat-containing domain superfamily / High mobility group box domain superfamily / GINS complex, subunit Psf3 superfamily / GINS, helical bundle-like domain superfamily / MCM OB domain / DNA replication complex GINS protein SLD5, C-terminal / Mini-chromosome maintenance protein / Mcm6, C-terminal winged-helix domain / GINS complex protein Sld5, alpha-helical domain / MCM, AAA-lid domain / MCM N-terminal domain
DNA replication licensing factor MCM2 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF1 / DNA replication licensing factor MCM6 / WD repeat and HMG-box DNA-binding protein 1 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM3 ...DNA replication licensing factor MCM2 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF1 / DNA replication licensing factor MCM6 / WD repeat and HMG-box DNA-binding protein 1 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM3 / Cell division control protein 45 homolog / DNA replication complex GINS protein PSF2
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.77 Å
AuthorsRzechorzek, N.J. / Pellegrini, L. / Chirgadze, D.Y. / Hardwick, S.W.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust104641/Z/14/Z United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2020
Title: CryoEM structures of human CMG-ATPγS-DNA and CMG-AND-1 complexes.
Authors: Neil J Rzechorzek / Steven W Hardwick / Vincentius A Jatikusumo / Dimitri Y Chirgadze / Luca Pellegrini /
Abstract: DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions ...DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one β-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJan 16, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 27, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 10, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 15, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
2: DNA replication licensing factor MCM2
3: DNA replication licensing factor MCM3
4: DNA replication licensing factor MCM4
5: DNA replication licensing factor MCM5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
A: DNA replication complex GINS protein PSF1
B: DNA replication complex GINS protein PSF2
C: DNA replication complex GINS protein PSF3
D: DNA replication complex GINS protein SLD5
E: Cell division control protein 45 homolog
F: WD repeat and HMG-box DNA-binding protein 1
G: WD repeat and HMG-box DNA-binding protein 1
H: WD repeat and HMG-box DNA-binding protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,104,14419
Polymers1,103,81714
Non-polymers3275
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area75650 Å2
ΔGint-354 kcal/mol
Surface area266680 Å2
MethodPISA

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Components

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DNA replication licensing factor ... , 6 types, 6 molecules 234567

#1: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2 homolog / Nuclear protein BM28


Mass: 102034.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM2, BM28, CCNL1, CDCL1, KIAA0030 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P49736, DNA helicase
#2: Protein DNA replication licensing factor MCM3 / DNA polymerase alpha holoenzyme-associated protein P1 / P1-MCM3 / RLF subunit beta / p102


Mass: 96043.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM3 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P25205, DNA helicase
#3: Protein DNA replication licensing factor MCM4 / CDC21 homolog / P1-CDC21


Mass: 96684.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM4, CDC21 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P33991, DNA helicase
#4: Protein DNA replication licensing factor MCM5 / CDC46 homolog / P1-CDC46


Mass: 82406.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM5, CDC46 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P33992, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / p105MCM


Mass: 93010.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM6 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q14566, DNA helicase
#6: Protein DNA replication licensing factor MCM7 / CDC47 homolog / P1.1-MCM3


Mass: 81411.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM7, CDC47, MCM2 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P33993, DNA helicase

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DNA replication complex GINS protein ... , 4 types, 4 molecules ABCD

#7: Protein DNA replication complex GINS protein PSF1 / GINS complex subunit 1


Mass: 23022.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS1, KIAA0186, PSF1 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q14691
#8: Protein DNA replication complex GINS protein PSF2 / GINS complex subunit 2


Mass: 21453.713 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS2, PSF2, CGI-122, DC5, HSPC037 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q9Y248
#9: Protein DNA replication complex GINS protein PSF3 / GINS complex subunit 3


Mass: 24562.611 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS3, PSF3 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q9BRX5
#10: Protein DNA replication complex GINS protein SLD5 / GINS complex subunit 4


Mass: 26081.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS4, SLD5 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q9BRT9

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Protein , 2 types, 4 molecules EFGH

#11: Protein Cell division control protein 45 homolog / PORC-PI-1


Mass: 65650.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC45, CDC45L, CDC45L2, UNQ374/PRO710 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: O75419
#12: Protein WD repeat and HMG-box DNA-binding protein 1 / Acidic nucleoplasmic DNA-binding protein 1 / And-1


Mass: 130484.750 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDHD1, AND1 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: O75717

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Non-polymers , 1 types, 5 molecules

#13: Chemical
ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Complex of human CMG bound to AND1COMPLEX#1-#120RECOMBINANT
2Human CMG helicaseCOMPLEX#1-#111RECOMBINANT
3AND1COMPLEX#121RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
33Homo sapiens (human)9606
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 54.3 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15393 / Symmetry type: POINT

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