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- EMDB-10619: CryoEM structure of human CMG bound to ATPgammaS and DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-10619
TitleCryoEM structure of human CMG bound to ATPgammaS and DNA
Map data
SampleComplex of human CMG bound to DNA
  • Human CMG helicase
  • DNA
  • (DNA replication licensing factor ...) x 6
  • (DNA replication complex GINS protein ...) x 4
  • Cell division control protein 45 homolog
  • nucleic-acidNucleic acid
  • (ligand) x 4
Function / homology
Function and homology information


DNA replication checkpoint signaling / mitotic DNA replication preinitiation complex assembly / DNA strand elongation involved in mitotic DNA replication / GINS complex / alpha DNA polymerase:primase complex / mitotic DNA replication / regulation of chromatin silencing at telomere / nuclear origin of replication recognition complex / cellular response to xenobiotic stimulus / CMG complex ...DNA replication checkpoint signaling / mitotic DNA replication preinitiation complex assembly / DNA strand elongation involved in mitotic DNA replication / GINS complex / alpha DNA polymerase:primase complex / mitotic DNA replication / regulation of chromatin silencing at telomere / nuclear origin of replication recognition complex / cellular response to xenobiotic stimulus / CMG complex / MCM complex / mitotic DNA replication initiation / negative regulation of DNA helicase activity / DNA replication preinitiation complex / double-strand break repair via break-induced replication / inner cell mass cell proliferation / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / regulation of phosphorylation / DNA strand elongation involved in DNA replication / cochlea development / DNA unwinding involved in DNA replication / DNA replication origin binding / ciliary basal body / cellular response to interleukin-4 / regulation of transcription involved in G1/S transition of mitotic cell cycle / cellular response to epidermal growth factor stimulus / DNA replication initiation / pre-replicative complex assembly / nucleosome assembly / single-stranded DNA binding / cell population proliferation / DNA helicase / DNA helicase activity / chromosome, telomeric region / histone binding / DNA replication / centrosome / chromatin / cellular response to DNA damage stimulus / response to drug / apoptotic process / chromatin binding / perinuclear region of cytoplasm / enzyme binding / DNA binding / membrane / nucleoplasm / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
GINS complex, subunit Psf1 / DNA replication licensing factor Mcm6 / GINS subunit, domain A / Mini-chromosome maintenance, conserved site / Nucleic acid-binding, OB-fold / DNA replication complex GINS protein Psf2 / GINS complex subunit Sld5 / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm5 / MCM N-terminal domain ...GINS complex, subunit Psf1 / DNA replication licensing factor Mcm6 / GINS subunit, domain A / Mini-chromosome maintenance, conserved site / Nucleic acid-binding, OB-fold / DNA replication complex GINS protein Psf2 / GINS complex subunit Sld5 / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm5 / MCM N-terminal domain / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm3 / DNA replication licensing factor Mcm2 / MCM domain / AAA+ ATPase domain / CDC45 family / P-loop containing nucleoside triphosphate hydrolase / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / Mini-chromosome maintenance protein / DNA replication complex GINS protein SLD5, C-terminal / MCM, AAA-lid domain / MCM OB domain / Mcm6, C-terminal winged-helix domain / GINS complex protein Sld5, alpha-helical domain / GINS, helical bundle-like domain superfamily
DNA replication licensing factor MCM6 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF1 / Cell division control protein 45 homolog / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM3 / DNA replication complex GINS protein PSF2
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsRzechorzek NJ / Pellegrini L
Funding support United Kingdom, 1 items
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 ReportSummary, Full report, XML, About validation report
History
DepositionJan 16, 2020-
Header (metadata) releaseJan 29, 2020-
Map releaseMay 27, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xtx
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10619.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 360 pix.
= 385.2 Å
1.07 Å/pix.
x 360 pix.
= 385.2 Å
1.07 Å/pix.
x 360 pix.
= 385.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.044788398 - 0.111151464
Average (Standard dev.)-0.00001422162 (±0.003039511)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 385.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z385.200385.200385.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0450.111-0.000

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Supplemental data

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Segmentation: #1

Fileemd_10619_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Human CMG helicase bound to DNA and ATP-gamma-S - refined half-map 1

Fileemd_10619_half_map_1.map
AnnotationHuman CMG helicase bound to DNA and ATP-gamma-S - refined half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Human CMG helicase bound to DNA and ATP-gamma-S - refined half-map 2

Fileemd_10619_half_map_2.map
AnnotationHuman CMG helicase bound to DNA and ATP-gamma-S - refined half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Complex of human CMG bound to DNA

EntireName: Complex of human CMG bound to DNA / Number of components: 19

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Component #1: protein, Complex of human CMG bound to DNA

ProteinName: Complex of human CMG bound to DNA / Recombinant expression: No

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Component #2: protein, Human CMG helicase

ProteinName: Human CMG helicase / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, DNA

ProteinName: DNA / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: protein, DNA replication licensing factor MCM2

ProteinName: DNA replication licensing factor MCM2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 102.034102 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, DNA replication licensing factor MCM3

ProteinName: DNA replication licensing factor MCM3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 96.04332 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #6: protein, DNA replication licensing factor MCM4

ProteinName: DNA replication licensing factor MCM4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 99.119461 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #7: protein, DNA replication licensing factor MCM5

ProteinName: DNA replication licensing factor MCM5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 82.406633 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #8: protein, DNA replication licensing factor MCM6

ProteinName: DNA replication licensing factor MCM6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 93.010273 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #9: protein, DNA replication licensing factor MCM7

ProteinName: DNA replication licensing factor MCM7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 81.411875 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #10: protein, DNA replication complex GINS protein PSF1

ProteinName: DNA replication complex GINS protein PSF1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 23.022469 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #11: protein, DNA replication complex GINS protein PSF2

ProteinName: DNA replication complex GINS protein PSF2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 25.336793 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #12: protein, DNA replication complex GINS protein PSF3

ProteinName: DNA replication complex GINS protein PSF3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 24.562611 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #13: protein, DNA replication complex GINS protein SLD5

ProteinName: DNA replication complex GINS protein SLD5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.081873 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #14: protein, Cell division control protein 45 homolog

ProteinName: Cell division control protein 45 homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 65.650555 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #15: nucleic-acid, DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')

nucleic acidName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3') / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DC)(DG)(DT)(DT)(DT)(DT)(DA)(DC)(DA)(DA) (DC)(DG)(DT)(DC)(DG)(DT)(DG)(DA)(DC)(DT) (DG)(DG)(DG)(DC)(DA)(DC)(DT)(DT)(DG)(DA) (DT)(DC)(DG)(DG)(DC)(DC)(DA)(DA)(DC)(DC) (DT)(DT)(DT)(DT)(DT)(DT) ...Sequence:
(DC)(DG)(DT)(DT)(DT)(DT)(DA)(DC)(DA)(DA) (DC)(DG)(DT)(DC)(DG)(DT)(DG)(DA)(DC)(DT) (DG)(DG)(DG)(DC)(DA)(DC)(DT)(DT)(DG)(DA) (DT)(DC)(DG)(DG)(DC)(DC)(DA)(DA)(DC)(DC) (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)
MassTheoretical: 21.390629 kDa
SourceSpecies: synthetic construct (others)

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Component #16: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #17: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Component #18: ligand, PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

LigandName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.523247 kDa

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Component #19: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 57.6 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 213527
3D reconstructionResolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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