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- EMDB-8540: Structural basis of MCM2-7 replicative helicase loading by ORC-Cd... -

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Basic information

Entry
Database: EMDB / ID: EMD-8540
TitleStructural basis of MCM2-7 replicative helicase loading by ORC-Cdc6 and Cdt1
Map data
SampleORC-Cdc6-Cdt1-MCM2-7
  • (DNA replication licensing factor ...) x 5
  • Minichromosome maintenance protein 5
  • (Cell division ...) x 2
  • (Origin recognition complex subunit ...) x 6
  • (nucleic-acidNucleic acid) x 2
  • ligand
Function / homology
Function and homology information


nuclear origin of replication recognition complex / MCM core complex / MCM complex binding / nuclear DNA replication / CMG complex / maintenance of rDNA / negative regulation of chromatin silencing at telomere / regulation of chromatin silencing at telomere / mitotic DNA replication / establishment of chromatin silencing ...nuclear origin of replication recognition complex / MCM core complex / MCM complex binding / nuclear DNA replication / CMG complex / maintenance of rDNA / negative regulation of chromatin silencing at telomere / regulation of chromatin silencing at telomere / mitotic DNA replication / establishment of chromatin silencing / regulation of DNA-dependent DNA replication initiation / negative regulation of DNA helicase activity / MCM complex / nuclear pre-replicative complex / DNA replication preinitiation complex / mitotic DNA replication initiation / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication checkpoint / single-stranded DNA helicase activity / double-strand break repair via break-induced replication / replication fork protection complex / nuclear replication fork / DNA strand elongation involved in DNA replication / chromatin silencing at silent mating-type cassette / nucleosome binding / histone H3-K4 methylation / DNA unwinding involved in DNA replication / DNA replication origin binding / DNA replication initiation / chromatin silencing at telomere / DNA helicase / DNA helicase activity / chromosome, telomeric region / G1/S transition of mitotic cell cycle / single-stranded DNA binding / chromosome / mitotic cell cycle / nuclear chromosome, telomeric region / ATPase activity / GTPase activity / cell division / GTP binding / cellular response to DNA damage stimulus / chromatin binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
Nucleic acid-binding, OB-fold / Origin recognition complex, subunit 6, fungi / Cdc6, C-terminal / Cell division protein Cdc6/18 / Bromo adjacent homology (BAH) domain / MCM domain / AAA+ ATPase domain / Mini-chromosome maintenance, conserved site / Origin recognition complex, subunit 1 / ATPase, AAA-type, core ...Nucleic acid-binding, OB-fold / Origin recognition complex, subunit 6, fungi / Cdc6, C-terminal / Cell division protein Cdc6/18 / Bromo adjacent homology (BAH) domain / MCM domain / AAA+ ATPase domain / Mini-chromosome maintenance, conserved site / Origin recognition complex, subunit 1 / ATPase, AAA-type, core / Origin recognition complex, subunit 2 / DNA replication licensing factor Mcm2 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm5 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex subunit 4 / Winged helix DNA-binding domain superfamily / MCM, AAA-lid domain / Origin recognition complex, subunit 6 / AAA lid domain / Mcm6, C-terminal winged-helix domain / ORC3, winged helix C-terminal / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm6 / Winged helix-like DNA-binding domain superfamily / MCM OB domain / Origin recognition complex subunit 4, C-terminal / Mini-chromosome maintenance protein / MCM N-terminal domain / P-loop containing nucleoside triphosphate hydrolase
Origin recognition complex subunit 6 / Origin recognition complex subunit 3 / Origin recognition complex subunit 1 / DNA replication licensing factor MCM6 / Origin recognition complex subunit 5 / Cell division cycle protein CDT1 / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM7 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM4 ...Origin recognition complex subunit 6 / Origin recognition complex subunit 3 / Origin recognition complex subunit 1 / DNA replication licensing factor MCM6 / Origin recognition complex subunit 5 / Cell division cycle protein CDT1 / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM7 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM4 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM3 / Cell division control protein 6 / Origin recognition complex subunit 4
Biological speciesSaccharomyces cerevisiae (baker's yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsYuan Z / Riera A / Bai L / Sun J / Spanos C / Chen ZA / Barbon M / Rappsilber J / Stillman B / Speck C / Li H
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2017
Title: Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1.
Authors: Zuanning Yuan / Alberto Riera / Lin Bai / Jingchuan Sun / Saikat Nandi / Christos Spanos / Zhuo Angel Chen / Marta Barbon / Juri Rappsilber / Bruce Stillman / Christian Speck / Huilin Li /
Abstract: To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus ...To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHDs) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.
Validation ReportPDB-ID: 5v8f

SummaryFull reportAbout validation report
History
DepositionDec 25, 2016-
Header (metadata) releaseFeb 22, 2017-
Map releaseFeb 22, 2017-
UpdateNov 8, 2017-
Current statusNov 8, 2017Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5v8f
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5v8f
  • Surface level: 0.021
  • 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_8540.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.01 Å/pix.
x 256 pix.
= 258.56 Å
1.01 Å/pix.
x 256 pix.
= 258.56 Å
1.01 Å/pix.
x 256 pix.
= 258.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.01 Å
Density
Contour LevelBy AUTHOR: 0.021 / Movie #1: 0.021
Minimum - Maximum-0.03851721 - 0.10812734
Average (Standard dev.)0.0004973546 (±0.004821395)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 258.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.011.011.01
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z258.560258.560258.560
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0390.1080.000

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Supplemental data

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Sample components

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Entire ORC-Cdc6-Cdt1-MCM2-7

EntireName: ORC-Cdc6-Cdt1-MCM2-7 / Number of components: 18

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Component #1: protein, ORC-Cdc6-Cdt1-MCM2-7

ProteinName: ORC-Cdc6-Cdt1-MCM2-7 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast) / Vector: pRS

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Component #2: protein, DNA replication licensing factor MCM2

ProteinName: DNA replication licensing factor MCM2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 98.911539 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, DNA replication licensing factor MCM3

ProteinName: DNA replication licensing factor MCM3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 107.653508 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #4: protein, DNA replication licensing factor MCM4

ProteinName: DNA replication licensing factor MCM4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 105.138375 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #5: protein, Minichromosome maintenance protein 5

ProteinName: Minichromosome maintenance protein 5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 86.505734 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #6: protein, DNA replication licensing factor MCM6

ProteinName: DNA replication licensing factor MCM6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 113.110211 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #7: protein, DNA replication licensing factor MCM7

ProteinName: DNA replication licensing factor MCM7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 95.049875 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #8: protein, Cell division cycle protein CDT1

ProteinName: Cell division cycle protein CDT1Cell cycle / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 68.486055 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #9: protein, Cell division control protein 6

ProteinName: Cell division control protein 6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 58.112367 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #10: protein, Origin recognition complex subunit 1

ProteinName: Origin recognition complex subunit 1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 104.546164 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #11: protein, Origin recognition complex subunit 2

ProteinName: Origin recognition complex subunit 2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 71.34218 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #12: protein, Origin recognition complex subunit 3

ProteinName: Origin recognition complex subunit 3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 72.161766 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #13: protein, Origin recognition complex subunit 4

ProteinName: Origin recognition complex subunit 4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 60.772152 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #14: protein, Origin recognition complex subunit 5

ProteinName: Origin recognition complex subunit 5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 55.347168 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #15: protein, Origin recognition complex subunit 6

ProteinName: Origin recognition complex subunit 6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 50.369531 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #16: nucleic-acid, DNA (39-MER)

nucleic acidName: DNA (39-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DA)(DT)(DT)(DT)(DA)(DA)(DG)(DT)(DA) (DT)(DT)(DG)(DT)(DT)(DT)(DG)(DT)(DG)(DC) (DA)(DC)(DT)(DT)(DG)(DC)(DC)(DT)(DG)(DC) (DA)(DG)(DG)(DC)(DC)(DT)(DT)(DT)(DT)
MassTheoretical: 11.967674 kDa
SourceSpecies: synthetic construct (others)

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Component #17: nucleic-acid, DNA (39-MER)

nucleic acidName: DNA (39-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DA)(DA)(DA)(DG)(DG)(DC)(DC)(DT)(DG) (DC)(DA)(DG)(DG)(DC)(DA)(DA)(DG)(DT)(DG) (DC)(DA)(DC)(DA)(DA)(DA)(DC)(DA)(DA)(DT) (DA)(DC)(DT)(DT)(DA)(DA)(DA)(DT)(DA)
MassTheoretical: 12.035815 kDa
SourceSpecies: synthetic construct (others)

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Component #18: ligand, PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

LigandName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 0.523247 kDa

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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: 50 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: 304288
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body
Output model

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