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- PDB-7v3u: Cryo-EM structure of MCM double hexamer with structured Mcm4-NSD -

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Basic information

Entry
Database: PDB / ID: 7v3u
TitleCryo-EM structure of MCM double hexamer with structured Mcm4-NSD
Components
  • (DNA replication licensing factor ...) x 5
  • Minichromosome maintenance protein 5
KeywordsCELL CYCLE / DNA replication initiation / Complex / Replicative helicase / Replication
Function / homology
Function and homology information


MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex ...MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / MCM complex / DNA replication preinitiation complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / replication fork protection complex / mitotic DNA replication initiation / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / subtelomeric heterochromatin formation / DNA replication initiation / heterochromatin formation / DNA helicase activity / helicase activity / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / metal ion binding / nucleus / 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 / Winged helix-like DNA-binding domain superfamily / 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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / 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
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsCheng, J. / Li, N. / Huo, Y. / Dang, S. / Tye, B. / Gao, N. / Zhai, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China) China
CitationJournal: Nat Commun / Year: 2022
Title: Structural Insight into the MCM double hexamer activation by Dbf4-Cdc7 kinase.
Authors: Jiaxuan Cheng / Ningning Li / Yunjing Huo / Shangyu Dang / Bik-Kwoon Tye / Ning Gao / Yuanliang Zhai /
Abstract: The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation by phosphorylation of the MCM double hexamer (MCM-DH) to promote helicase activation. Here, we determine a series of cryo ...The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation by phosphorylation of the MCM double hexamer (MCM-DH) to promote helicase activation. Here, we determine a series of cryo electron microscopy (cryo-EM) structures of yeast DDK bound to the MCM-DH. These structures, occupied by one or two DDKs, differ primarily in the conformations of the kinase core. The interactions of DDK with the MCM-DH are mediated exclusively by subunit Dbf4 straddling across the hexamer interface on the three N-terminal domains (NTDs) of subunits Mcm2, Mcm6, and Mcm4. This arrangement brings Cdc7 close to its only essential substrate, the N-terminal serine/threonine-rich domain (NSD) of Mcm4. Dbf4 further displaces the NSD from its binding site on Mcm4-NTD, facilitating an immediate targeting of this motif by Cdc7. Moreover, the active center of Cdc7 is occupied by a unique Dbf4 inhibitory loop, which is disengaged when the kinase core assumes wobbling conformations. This study elucidates the versatility of Dbf4 in regulating the ordered multisite phosphorylation of the MCM-DH by Cdc7 kinase during helicase activation.
History
DepositionAug 11, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 13, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
2: DNA replication licensing factor MCM2
3: DNA replication licensing factor MCM3
4: DNA replication licensing factor MCM4
5: Minichromosome maintenance protein 5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
B: DNA replication licensing factor MCM2
C: DNA replication licensing factor MCM3
D: DNA replication licensing factor MCM4
E: Minichromosome maintenance protein 5
F: DNA replication licensing factor MCM6
G: DNA replication licensing factor MCM7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,219,77146
Polymers1,212,73812
Non-polymers7,03334
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA replication licensing factor ... , 5 types, 10 molecules 2B3C4D6F7G

#1: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2


Mass: 98911.539 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P29469, DNA helicase
#2: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 107653.508 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P24279, DNA helicase
#3: Protein DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 105138.375 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P30665, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P53091, DNA helicase
#6: Protein DNA replication licensing factor MCM7 / Cell division control protein 47 / Minichromosome maintenance protein 7


Mass: 95049.875 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P38132, DNA helicase

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Protein , 1 types, 2 molecules 5E

#4: Protein Minichromosome maintenance protein 5 / / Cell division control protein 46


Mass: 86505.734 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P29496, DNA helicase

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Non-polymers , 4 types, 34 molecules

#7: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MCM double hexamer (DH) / Type: COMPLEX / Entity ID: #1-#6 / Source: NATURAL
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: S288c
Buffer solutionpH: 7.6
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
EM embeddingMaterial: Ice
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 46 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00463631
ELECTRON MICROSCOPYf_angle_d0.66686109
ELECTRON MICROSCOPYf_dihedral_angle_d6.378743
ELECTRON MICROSCOPYf_chiral_restr0.0479991
ELECTRON MICROSCOPYf_plane_restr0.00611074

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