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Yorodumi- EMDB-13621: Structure of MCM2-7 DH complexed with Cdc7-Dbf4 in the presence o... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13621 | |||||||||||||||
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Title | Structure of MCM2-7 DH complexed with Cdc7-Dbf4 in the presence of ATPgS, state III (B1 map) | |||||||||||||||
Map data | Body 1 of multi-body refinement of MD-(ATPgS) state III | |||||||||||||||
Sample |
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Keywords | Helicase / Activation / Kinase / Phosphorylation / REPLICATION | |||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||||||||
Authors | Saleh A / Noguchi Y / Aramayo R / Ivanova ME / Speck C | |||||||||||||||
Funding support | United Kingdom, 4 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_13621.map.gz | 138.4 MB | EMDB map data format | |
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Header (meta data) | emd-13621-v30.xml emd-13621.xml | 13 KB 13 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13621_fsc.xml | 12.8 KB | Display | FSC data file |
Images | emd_13621.png | 59.5 KB | ||
Masks | emd_13621_msk_1.map | 178 MB | Mask map | |
Filedesc metadata | emd-13621.cif.gz | 4.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13621 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13621 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13621.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Body 1 of multi-body refinement of MD-(ATPgS) state III | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_13621_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4
Entire | Name: MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4 |
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Components |
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-Supramolecule #1: MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4
Supramolecule | Name: MCM2-7 double hexamer bound to two copies of Cdc7-Dbf4 type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Molecular weight | Theoretical: 1.5 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: Quantifoil R2/2 / 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 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 1.5 seconds and blot force +2. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number real images: 9909 / Average electron dose: 45.9 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |