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- PDB-6xtx: CryoEM structure of human CMG bound to ATPgammaS and DNA -

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Basic information

Entry
Database: PDB / ID: 6xtx
TitleCryoEM structure of human CMG bound to ATPgammaS and DNA
Components
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 6
  • Cell division control protein 45 homolog
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
KeywordsREPLICATION / CMG / Helicase / ATPase / Replisome
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 / mitotic DNA replication / alpha DNA polymerase:primase complex / CMG complex / 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 / mitotic DNA replication / alpha DNA polymerase:primase complex / CMG complex / DNA replication checkpoint signaling / MCM complex / regulation of phosphorylation / DNA replication preinitiation complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / inner cell mass cell proliferation / G1/S-Specific Transcription / cochlea development / DNA unwinding involved in DNA replication / DNA replication origin binding / DNA replication initiation / Activation of the pre-replicative complex / Activation of ATR in response to replication stress / DNA helicase activity / cellular response to interleukin-4 / ciliary basal body / helicase activity / Assembly of the pre-replicative complex / nucleosome assembly / Orc1 removal from chromatin / cellular response to xenobiotic stimulus / single-stranded DNA binding / histone binding / DNA helicase / cell population proliferation / DNA replication / chromosome, telomeric region / cell cycle / centrosome / apoptotic process / DNA damage response / chromatin binding / chromatin / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / membrane / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ribosomal Protein L9; domain 1 - #60 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #1030 / Ribosomal Protein L9; domain 1 - #50 / Ribosomal Protein L9; domain 1 / CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily ...Ribosomal Protein L9; domain 1 - #60 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #1030 / Ribosomal Protein L9; domain 1 - #50 / Ribosomal Protein L9; domain 1 / CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain 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 / 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 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / Up-down Bundle / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / 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 ...ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / 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 speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsRzechorzek, N.J. / Pellegrini, L.
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.
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
M: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)742,89525
Polymers740,07112
Non-polymers2,82413
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area71600 Å2
ΔGint-376 kcal/mol
Surface area224370 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: 99119.461 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: 25336.793 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 / DNA chain , 2 types, 2 molecules EM

#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: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 21390.629 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 13 molecules

#13: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn
#14: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#15: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#16: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg

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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 DNACOMPLEX#1-#120MULTIPLE SOURCES
2Human CMG helicaseCOMPLEX#1-#111RECOMBINANT
3DNACOMPLEX#121RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
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: 57.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (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: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213527 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00941569
ELECTRON MICROSCOPYf_angle_d0.73756167
ELECTRON MICROSCOPYf_dihedral_angle_d8.53425610
ELECTRON MICROSCOPYf_chiral_restr0.0496308
ELECTRON MICROSCOPYf_plane_restr0.0047270

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