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- EMDB-13657: Structure of double-stranded DNA-bound MCM2-7 DH complexed with C... -

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Basic information

Entry
Database: EMDB / ID: EMD-13657
TitleStructure of double-stranded DNA-bound MCM2-7 DH complexed with Cdc7-Dbf4 in the presence of ATP (B1 map)
Map dataBody 1 of multi-body refinement of MD-(ATP)
Sample
  • Complex: MCM2-7 double hexamer bound to double-stranded DNA and two copies of Cdc7-Dbf4
KeywordsHelicase / Activation / Kinase / Phosphorylation / REPLICATION
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.3 Å
AuthorsSaleh A / Noguchi Y / Aramayo R / Ivanova ME / Speck C
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T005378/1 United Kingdom
Wellcome Trust107903/Z/15/Z United Kingdom
Medical Research Council (MRC, United Kingdom)A652-5PY40 United Kingdom
Wellcome Trust206175/Z/17/Z United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer.
Authors: Almutasem Saleh / Yasunori Noguchi / Ricardo Aramayo / Marina E Ivanova / Kathryn M Stevens / Alex Montoya / S Sunidhi / Nicolas Lopez Carranza / Marcin J Skwark / Christian Speck /
Abstract: The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at ...The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.
History
DepositionSep 28, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13657.png

Downloads & links

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Map

FileDownload / File: emd_13657.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBody 1 of multi-body refinement of MD-(ATP)
Voxel sizeX=Y=Z: 1.085 Å
Density
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.026934834 - 0.05595885
Average (Standard dev.)-0.0009898383 (±0.0049569006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 390.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13657_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Post-processed map of Body 1 of multi-body refinement...

Fileemd_13657_additional_1.map
AnnotationPost-processed map of Body 1 of multi-body refinement of MD-(ATP) (B-factor of -100 applied)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MCM2-7 double hexamer bound to double-stranded DNA and two copies...

EntireName: MCM2-7 double hexamer bound to double-stranded DNA and two copies of Cdc7-Dbf4
Components
  • Complex: MCM2-7 double hexamer bound to double-stranded DNA and two copies of Cdc7-Dbf4

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Supramolecule #1: MCM2-7 double hexamer bound to double-stranded DNA and two copies...

SupramoleculeName: MCM2-7 double hexamer bound to double-stranded DNA and two copies of Cdc7-Dbf4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 1.54 MDa

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 1.5 seconds and blot force +2.

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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 / Cs: 2.7 mm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 3416 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: An ab initio initial model was generated using RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 5528
FSC plot (resolution estimation)

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