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- PDB-5bk4: Cryo-EM structure of Mcm2-7 double hexamer on dsDNA -

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Basic information

Entry
Database: PDB / ID: 5bk4
TitleCryo-EM structure of Mcm2-7 double hexamer on dsDNA
Components
  • (DNA (60-mer), strand ...) x 2
  • (DNA replication licensing factor ...) x 6
KeywordsHYDROLASE/DNA / complex / DNA replication / HYDROLASE-DNA complex
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 / DNA replication preinitiation complex / nuclear pre-replicative complex / regulation of DNA-dependent DNA replication initiation / 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
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single 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 Report
SummaryFull reportAbout validation report
DateDeposition: Sep 12, 2017 / Release: Oct 25, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Oct 25, 2017Structure modelrepositoryInitial release
1.1Nov 8, 2017Structure modelDatabase referencescitation_citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title
1.2Nov 22, 2017Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last
1.3Dec 6, 2017Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization

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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: DNA replication licensing factor MCM5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
A: DNA replication licensing factor MCM2
B: DNA replication licensing factor MCM3
C: DNA replication licensing factor MCM4
D: DNA replication licensing factor MCM5
E: DNA replication licensing factor MCM6
F: DNA replication licensing factor MCM7
S: DNA (60-mer), strand 1
O: DNA (60-mer), strand 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,253,14022
Polyers1,249,72214
Non-polymers3,4188
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)115650
ΔGint (kcal/M)-582
Surface area (Å2)387600

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Components

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DNA replication licensing factor ... , 6 types, 12 molecules 2A3B4C5D6E7F

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


Mass: 98911.539 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P29469, DNA helicase
#2: Protein/peptide DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 107653.508 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P24279, DNA helicase
#3: Protein/peptide DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 105138.375 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P30665, DNA helicase
#4: Protein/peptide DNA replication licensing factor MCM5 / Minichromosome maintenance protein 5 / Cell division control protein 46


Mass: 86505.734 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P29496, DNA helicase
#5: Protein/peptide DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P53091, DNA helicase
#6: Protein/peptide DNA replication licensing factor MCM7 / Cell division control protein 47 / Minichromosome maintenance protein 7


Mass: 95049.875 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P38132, DNA helicase

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DNA (60-mer), strand ... , 2 types, 2 molecules SO

#7: DNA chain DNA (60-mer), strand 1


Mass: 18491.848 Da / Num. of mol.: 1 / Source: (synth.) Saccharomyces cerevisiae (baker's yeast)
#8: DNA chain DNA (60-mer), strand 2


Mass: 18491.848 Da / Num. of mol.: 1 / Source: (synth.) Saccharomyces cerevisiae (baker's yeast)

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Non-polymers , 1 types, 8 molecules

#9: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 8 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1Mcm double hexamer bound to double-stranded DNACOMPLEX1,2,3,4,5,6,7,80MULTIPLE SOURCES
2Mcm double hexamerCOMPLEX1,2,3,4,5,61RECOMBINANT
3synthetic double-stranded DNACOMPLEX7,81NATURAL
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
114932Saccharomyces cerevisiae (baker's yeast)
224932Saccharomyces cerevisiae (baker's yeast)
334932Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 10 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2particle selection
2RELION1.4particle selection
3RELION2.0image acquisition
4RELION1.4image acquisition
6CTFFIND4CTF correction
7GctfCTF correction
10chimera1.11model fitting
11Coot0.8model fitting
13PHENIX1.11model refinement
14RELION2initial Euler assignment
15RELION2final Euler assignment
17RELION23D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 58772 / Symmetry type: POINT
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00863728
ELECTRON MICROSCOPYf_angle_d1.21586740
ELECTRON MICROSCOPYf_dihedral_angle_d14.06838738
ELECTRON MICROSCOPYf_chiral_restr0.06310082
ELECTRON MICROSCOPYf_plane_restr0.00710746

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