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- EMDB-9400: Cryo-EM structure of Mcm2-7 double hexamer on dsDNA -

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Basic information

Entry
Database: EMDB / ID: 9400
TitleCryo-EM structure of Mcm2-7 double hexamer on dsDNA
Map dataMcm2-7 double hexamer on dsDNA
SampleMcm double hexamer bound to double-stranded DNA
  • Mcm double hexamer
  • synthetic double-stranded DNA
  • (DNA replication licensing factor ...) x 6
  • (nucleic-acidNucleic acid) x 2
  • ligand
Function / homologyP-loop containing nucleoside triphosphate hydrolase / Mini-chromosome maintenance protein / MCM domain / MCM OB domain / AAA+ ATPase domain / 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 ...P-loop containing nucleoside triphosphate hydrolase / Mini-chromosome maintenance protein / MCM domain / MCM OB domain / AAA+ ATPase domain / 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 / MCM2/3/5 family / DNA replication licensing factor Mcm7 / Mini-chromosome maintenance protein 2 / MCM N-terminal domain / MCM OB domain / MCM family signature. / MCM family domain profile. / Nucleic acid-binding, OB-fold / Orc1 removal from chromatin / Mini-chromosome maintenance, conserved site / Activation of the pre-replicative complex / Switching of origins to a post-replicative state / MCM N-terminal domain / MCM complex binding / negative regulation of ATP-dependent DNA helicase activity / MCM core complex / CMG complex / nuclear DNA replication / mitotic DNA replication initiation / negative regulation of chromatin silencing at telomere / MCM complex / establishment of chromatin silencing / regulation of DNA-dependent DNA replication initiation / DNA replication preinitiation complex / nuclear pre-replicative complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / single-stranded DNA-dependent ATPase activity / double-strand break repair via break-induced replication / nuclear replication fork / DNA strand elongation involved in DNA replication / replication fork protection complex / DNA unwinding involved in DNA replication / DNA duplex unwinding / chromatin silencing at silent mating-type cassette / DNA helicase activity / DNA replication origin binding / chromatin silencing at telomere / DNA replication initiation / DNA helicase / helicase activity / chromosome, telomeric region / single-stranded DNA binding / nuclear chromosome, telomeric region / cellular response to DNA damage stimulus / chromatin binding / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM6
Function and homology information
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsLi H / Yuan Z / Bai L
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2017
Title: Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model.
Authors: Yasunori Noguchi / Zuanning Yuan / Lin Bai / Sarah Schneider / Gongpu Zhao / Bruce Stillman / Christian Speck / Huilin Li
Abstract: During replication initiation, the core component of the helicase-the Mcm2-7 hexamer-is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked ...During replication initiation, the core component of the helicase-the Mcm2-7 hexamer-is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked axial channel. How the origin DNA interacts with the axial channel is not understood, but the interaction could provide key insights into Mcm2-7 function and regulation. Here, we report the cryo-EM structure of the Mcm2-7 DH on dsDNA and show that the DNA is zigzagged inside the central channel. Several of the Mcm subunit DNA-binding loops, such as the oligosaccharide-oligonucleotide loops, helix 2 insertion loops, and presensor 1 (PS1) loops, are well defined, and many of them interact extensively with the DNA. The PS1 loops of Mcm 3, 4, 6, and 7, but not 2 and 5, engage the lagging strand with an approximate step size of one base per subunit. Staggered coupling of the two opposing hexamers positions the DNA right in front of the two Mcm2-Mcm5 gates, with each strand being pressed against one gate. The architecture suggests that lagging-strand extrusion initiates in the middle of the DH that is composed of the zinc finger domains of both hexamers. To convert the Mcm2-7 DH structure into the Mcm2-7 hexamer structure found in the active helicase, the N-tier ring of the Mcm2-7 hexamer in the DH-dsDNA needs to tilt and shift laterally. We suggest that these N-tier ring movements cause the DNA strand separation and lagging-strand extrusion.
Validation ReportPDB-ID: 5bk4

SummaryFull reportAbout validation report
DateDeposition: Sep 12, 2017 / Header (metadata) release: Oct 25, 2017 / Map release: Oct 25, 2017 / Last update: Jul 18, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.026
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.026
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-5bk4
  • Surface level: 0.026
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9400.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.09 Å/pix.
= 278.528 Å
256 pix
1.09 Å/pix.
= 278.528 Å
256 pix
1.09 Å/pix.
= 278.528 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.088 Å
Density
Contour Level:0.026 (by author), 0.026 (movie #1):
Minimum - Maximum-0.13268049 - 0.2544556
Average (Standard dev.)0.001590641 (0.008777504)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.0.0.
Limit255.255.255.
Spacing256256256
CellA=B=C: 278.528 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0881.0881.088
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z278.528278.528278.528
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1330.2540.002

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Supplemental data

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Sample components

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Entire Mcm double hexamer bound to double-stranded DNA

EntireName: Mcm double hexamer bound to double-stranded DNA / Number of components: 12

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Component #1: protein, Mcm double hexamer bound to double-stranded DNA

ProteinName: Mcm double hexamer bound to double-stranded DNA / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, Mcm double hexamer

ProteinName: Mcm double hexamer / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, synthetic double-stranded DNA

ProteinName: synthetic double-stranded DNA / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #4: protein, DNA replication licensing factor MCM2

ProteinName: DNA replication licensing factor MCM2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 98.911539 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #5: protein, DNA replication licensing factor MCM3

ProteinName: DNA replication licensing factor MCM3 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 107.653508 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #6: protein, DNA replication licensing factor MCM4

ProteinName: DNA replication licensing factor MCM4 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 105.138375 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #7: protein, DNA replication licensing factor MCM5

ProteinName: DNA replication licensing factor MCM5 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 86.505734 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #8: protein, DNA replication licensing factor MCM6

ProteinName: DNA replication licensing factor MCM6 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 113.110211 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #9: protein, DNA replication licensing factor MCM7

ProteinName: DNA replication licensing factor MCM7 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 95.049875 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #10: nucleic-acid, DNA (60-mer), strand 1

Nucleic-acidName: DNA (60-mer), strand 1 / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA) (DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT) (DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA) (DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT) (DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA) (DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)
MassTheoretical: 18.491848 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #11: nucleic-acid, DNA (60-mer), strand 2

Nucleic-acidName: DNA (60-mer), strand 2 / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC) (DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG) (DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC) (DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG) (DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC) (DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG)
MassTheoretical: 18.491848 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #12: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 8
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: 1 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 / Number of projections: 58772
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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