+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0723 | ||||||||||||||||||||||||
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Title | PolD-PCNA-DNA (form A) | ||||||||||||||||||||||||
Map data | |||||||||||||||||||||||||
Sample |
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Function / homology | Function and homology information : / : / DNA polymerase complex / exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intron homing / intein-mediated protein splicing / DNA catabolic process / DNA strand elongation involved in DNA replication / leading strand elongation ...: / : / DNA polymerase complex / exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intron homing / intein-mediated protein splicing / DNA catabolic process / DNA strand elongation involved in DNA replication / leading strand elongation / DNA polymerase processivity factor activity / regulation of DNA replication / 3'-5' exonuclease activity / DNA-templated DNA replication / endonuclease activity / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding / identical protein binding Similarity search - Function | ||||||||||||||||||||||||
Biological species | Thermococcus kodakarensis (archaea) / synthetic construct (others) | ||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.9 Å | ||||||||||||||||||||||||
Authors | Mayanagi K / Oki K / Miyazaki N / Ishino S / Yamagami T / Iwasaki K / Kohda D / Morikawa K / Shirai T / Ishino Y | ||||||||||||||||||||||||
Funding support | Japan, 7 items
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Citation | Journal: BMC Biol / Year: 2020 Title: Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy. Authors: Kouta Mayanagi / Keisuke Oki / Naoyuki Miyazaki / Sonoko Ishino / Takeshi Yamagami / Kosuke Morikawa / Kenji Iwasaki / Daisuke Kohda / Tsuyoshi Shirai / Yoshizumi Ishino / Abstract: BACKGROUND: DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA ...BACKGROUND: DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3'-5' editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM). RESULTS: Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus ...RESULTS: Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro. CONCLUSIONS: Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for ...CONCLUSIONS: Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life. | ||||||||||||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_0723.map.gz | 14.3 MB | EMDB map data format | |
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Header (meta data) | emd-0723-v30.xml emd-0723.xml | 13.7 KB 13.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_0723_fsc.xml | 5.8 KB | Display | FSC data file |
Images | emd_0723.png | 272.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0723 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0723 | HTTPS FTP |
-Related structure data
Related structure data | 6knbMC 0725C 6kncC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_0723.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.12 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Thermococcus kodakarensis PolD-PCNA-DNA
Entire | Name: Thermococcus kodakarensis PolD-PCNA-DNA |
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Components |
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-Supramolecule #1: Thermococcus kodakarensis PolD-PCNA-DNA
Supramolecule | Name: Thermococcus kodakarensis PolD-PCNA-DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#6 |
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-Supramolecule #2: Thermococcus kodakarensis Pol D DP1 subunit
Supramolecule | Name: Thermococcus kodakarensis Pol D DP1 subunit / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: Thermococcus kodakarensis (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Supramolecule #3: Thermococcus kodakarensis Pol D DP2 subunit
Supramolecule | Name: Thermococcus kodakarensis Pol D DP2 subunit / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: Thermococcus kodakarensis (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Supramolecule #4: Thermococcus kodakarensis PCNA trimer ring
Supramolecule | Name: Thermococcus kodakarensis PCNA trimer ring / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4 |
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Source (natural) | Organism: Thermococcus kodakarensis (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Supramolecule #5: pri25DNA
Supramolecule | Name: pri25DNA / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #5 Details: Sequence:(DC)(DG)(DA)(DA)(DC)(DT)(DG)(DC)(DC)(DT)(DG)(DG)(DA)(DA)(DT)(DC)(DC)(DT)(DG)(DA)(DC)(DG)(DA)(DC)(DA) |
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Source (natural) | Organism: synthetic construct (others) |
Recombinant expression | Organism: synthetic construct (others) |
-Supramolecule #6: temp35DNA
Supramolecule | Name: temp35DNA / type: complex / ID: 6 / Parent: 1 / Macromolecule list: #6 Details: Sequence: (DG)(DT)(DT)(DC)(DG)(DC)(DT)(DA)(DC)(DA)(DT)(DG)(DT)(DC)(DG)(DT)(DC)(DA)(DG)(DG)(DA)(DT)(DT)(DC)(DC)(DA)(DG)(DG)(DC)(DA)(DG)(DT)(DT)(DC)(DG) |
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Source (natural) | Organism: synthetic construct (others) |
Recombinant expression | Organism: synthetic construct (others) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 |
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy |
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |