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- EMDB-10620: CryoEM structure of human CMG bound to ATPgammaS and DNA - MCM2-7... -

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Basic information

Entry
Database: EMDB / ID: EMD-10620
TitleCryoEM structure of human CMG bound to ATPgammaS and DNA - MCM2-7 C-tier
Map dataHuman CMG helicase bound to DNA and ATPgammaS - final postprocessed map for Ctier only, moved to Ctier centre of mass
Sample
  • Complex: Complex of human CMG bound to DNA
    • Complex: Human CMG helicase - MCM2-7 Ctier only
Function / homology
Function and homology information


Switching of origins to a post-replicative state / Unwinding of DNA / nuclear origin of replication recognition complex / mitotic DNA replication / alpha DNA polymerase:primase complex / CMG complex / MCM complex / regulation of phosphorylation / double-strand break repair via break-induced replication / mitotic DNA replication initiation ...Switching of origins to a post-replicative state / Unwinding of DNA / nuclear origin of replication recognition complex / mitotic DNA replication / alpha DNA polymerase:primase complex / CMG complex / MCM complex / regulation of phosphorylation / 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 / 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 / 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 / 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
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 ...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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
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
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.41 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.
History
DepositionJan 16, 2020-
Header (metadata) releaseJan 29, 2020-
Map releaseMay 27, 2020-
UpdateJul 15, 2020-
Current statusJul 15, 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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10620.png

Downloads & links

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Map

FileDownload / File: emd_10620.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman CMG helicase bound to DNA and ATPgammaS - final postprocessed map for Ctier only, moved to Ctier centre of mass
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.023375656 - 0.08685699
Average (Standard dev.)-0.00013239747 (±0.0021073332)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0460.127-0.000

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Supplemental data

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Mask #1

Fileemd_10620_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Human CMG helicase bound to DNA and ATPgammaS...

Fileemd_10620_half_map_1.map
AnnotationHuman CMG helicase bound to DNA and ATPgammaS - refined half-map 1 for Ctier only, moved to Ctier centre of mass
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Human CMG helicase bound to DNA and ATPgammaS...

Fileemd_10620_half_map_2.map
AnnotationHuman CMG helicase bound to DNA and ATPgammaS - refined half-map 2 for Ctier only, moved to Ctier centre of mass
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Components
  • Complex: Complex of human CMG bound to DNA
    • Complex: Human CMG helicase - MCM2-7 Ctier only

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Supramolecule #1: Complex of human CMG bound to DNA

SupramoleculeName: Complex of human CMG bound to DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#12
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #2: Human CMG helicase - MCM2-7 Ctier only

SupramoleculeName: Human CMG helicase - MCM2-7 Ctier only / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#11
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 57.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 181401
FSC plot (resolution estimation)

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