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- EMDB-10621: CryoEM structure of human CMG bound to AND-1 (CMGA) -

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Basic information

Entry
Database: EMDB / ID: EMD-10621
TitleCryoEM structure of human CMG bound to AND-1 (CMGA)
Map data
SampleComplex of human CMG bound to AND1
  • Human CMG helicase
  • AND1
  • (DNA replication licensing factor ...) x 6
  • (DNA replication complex GINS protein ...) x 4
  • Cell division control protein 45 homolog
  • WD repeat and HMG-box DNA-binding protein 1
  • ligand
Function / homology
Function and homology information


DNA replication checkpoint signaling / mitotic DNA replication preinitiation complex assembly / GINS complex / DNA strand elongation involved in mitotic DNA replication / alpha DNA polymerase:primase complex / mitotic DNA replication / regulation of chromatin silencing at telomere / CMG complex / cellular response to xenobiotic stimulus / nuclear origin of replication recognition complex ...DNA replication checkpoint signaling / mitotic DNA replication preinitiation complex assembly / GINS complex / DNA strand elongation involved in mitotic DNA replication / alpha DNA polymerase:primase complex / mitotic DNA replication / regulation of chromatin silencing at telomere / CMG complex / cellular response to xenobiotic stimulus / nuclear origin of replication recognition complex / 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 / double-strand break repair via break-induced replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / regulation of phosphorylation / 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 / ciliary basal body / 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-dependent DNA replication / G1/S transition of mitotic cell cycle / single-stranded DNA binding / cell population proliferation / DNA helicase / DNA helicase activity / mitotic cell cycle / chromosome, telomeric region => GO:0000781 / histone binding / DNA replication / centrosome / DNA repair / 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
DNA replication licensing factor Mcm7 / MCM domain / Minichromosome loss protein Mcl1, middle region / GINS subunit, domain A / WD40 repeat, conserved site / GINS complex subunit Sld5 / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / Nucleic acid-binding, OB-fold / GINS complex, subunit Psf3 ...DNA replication licensing factor Mcm7 / MCM domain / Minichromosome loss protein Mcl1, middle region / GINS subunit, domain A / WD40 repeat, conserved site / GINS complex subunit Sld5 / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / Nucleic acid-binding, OB-fold / GINS complex, subunit Psf3 / High mobility group box domain / WD40 repeat / P-loop containing nucleoside triphosphate hydrolase / AAA+ ATPase domain / CDC45 family / GINS complex, subunit Psf1 / DNA replication complex GINS protein Psf2 / DNA replication licensing factor Mcm2 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm6 / Anaphase-promoting complex subunit 4, WD40 domain / Mini-chromosome maintenance, conserved site / MCM N-terminal domain / GINS complex, subunit Psf3 superfamily / WD40-repeat-containing domain superfamily / MCM OB domain / DNA replication complex GINS protein SLD5, C-terminal / High mobility group box domain superfamily / Mini-chromosome maintenance protein / GINS complex protein Sld5, alpha-helical domain / Mcm6, C-terminal winged-helix domain / MCM, AAA-lid domain / GINS, helical bundle-like domain superfamily
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.77 Å
AuthorsRzechorzek NJ / Pellegrini L / Chirgadze DY / Hardwick SW
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: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xty
  • Surface level: 0.4
  • 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_10621.map.gz / Format: CCP4 / Size: 303.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 430 pix.
= 455.8 Å
1.06 Å/pix.
x 430 pix.
= 455.8 Å
1.06 Å/pix.
x 430 pix.
= 455.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-0.5425978 - 1.6387757
Average (Standard dev.)0.0029354305 (±0.069991335)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions430430430
Spacing430430430
CellA=B=C: 455.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z430430430
origin x/y/z0.0000.0000.000
length x/y/z455.800455.800455.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS430430430
D min/max/mean-0.5431.6390.003

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Supplemental data

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Segmentation: #1

Fileemd_10621_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Human CMG helicase bound to AND-1 in the...

Fileemd_10621_additional.map
AnnotationHuman CMG helicase bound to AND-1 in the presence of ATPgammaS - mask used for refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Human CMG helicase bound to AND-1 in the...

Fileemd_10621_half_map_1.map
AnnotationHuman CMG helicase bound to AND-1 in the presence of ATPgammaS - refined half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Human CMG helicase bound to AND-1 in the...

Fileemd_10621_half_map_2.map
AnnotationHuman CMG helicase bound to AND-1 in the presence of ATPgammaS - 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 AND1

EntireName: Complex of human CMG bound to AND1 / Number of components: 16

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Component #1: protein, Complex of human CMG bound to AND1

ProteinName: Complex of human CMG bound to AND1 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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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, AND1

ProteinName: AND1 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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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: 96.684852 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: 21.453713 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: protein, WD repeat and HMG-box DNA-binding protein 1

ProteinName: WD repeat and HMG-box DNA-binding protein 1 / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 130.48475 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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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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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: 54.3 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 15393
3D reconstructionResolution: 6.77 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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