[English] 日本語
Yorodumi- EMDB-1618: 3D reconstruction of heterodimeric yeast Pol alpha using electron... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1618 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | 3D reconstruction of heterodimeric yeast Pol alpha using electron microscopy | |||||||||
Map data | 3D reconstruction of heterodimeric yeast Pol alpha using electron microscopy | |||||||||
Sample |
| |||||||||
Keywords | DNA replication / DNA Polymerase alpha / Electron microscopy | |||||||||
Function / homology | Function and homology information protein binding / RNA-templated DNA biosynthetic process / premeiotic DNA replication / nucleoside binding / alpha DNA polymerase:primase complex / lagging strand elongation / telomere capping / DNA synthesis involved in DNA repair / DNA replication initiation / nuclear envelope ...protein binding / RNA-templated DNA biosynthetic process / premeiotic DNA replication / nucleoside binding / alpha DNA polymerase:primase complex / lagging strand elongation / telomere capping / DNA synthesis involved in DNA repair / DNA replication initiation / nuclear envelope / DNA-directed DNA polymerase activity / nucleotide binding / mitochondrion / DNA binding Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 22.9 Å | |||||||||
Authors | Klinge S / Nunez-Ramirez R / Llorca O / Pellegrini L | |||||||||
Citation | Journal: EMBO J / Year: 2009 Title: 3D architecture of DNA Pol alpha reveals the functional core of multi-subunit replicative polymerases. Authors: Sebastian Klinge / Rafael Núñez-Ramírez / Oscar Llorca / Luca Pellegrini / Abstract: Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol alpha, Pol delta and Pol epsilon. The conserved catalytic and regulatory B subunits associate in ...Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol alpha, Pol delta and Pol epsilon. The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol alpha. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol alpha reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B-CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases. | |||||||||
History |
|
-Structure visualization
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
---|---|
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1618.map.gz | 68.5 KB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-1618-v30.xml emd-1618.xml | 13.7 KB 13.7 KB | Display Display | EMDB header |
Images | EMD-1618.tif emd_1618.tif | 1.6 MB 1.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1618 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1618 | HTTPS FTP |
-Validation report
Summary document | emd_1618_validation.pdf.gz | 193.7 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_1618_full_validation.pdf.gz | 192.9 KB | Display | |
Data in XML | emd_1618_validation.xml.gz | 4.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1618 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1618 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|
-Map
File | Download / File: emd_1618.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | 3D reconstruction of heterodimeric yeast Pol alpha using electron microscopy | ||||||||||||||||||||
Voxel size | X=Y=Z: 3.8 Å | ||||||||||||||||||||
Density |
| ||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Sample components
-Entire : Yeast DNA polymerase alpha
Entire | Name: Yeast DNA polymerase alpha |
---|---|
Components |
|
-Supramolecule #1000: Yeast DNA polymerase alpha
Supramolecule | Name: Yeast DNA polymerase alpha / type: sample / ID: 1000 Oligomeric state: One monomer of DNA polymerase alpha binds to a monomer of B-subunit Number unique components: 2 |
---|---|
Molecular weight | Theoretical: 185 KDa / Method: Gel Filtration |
-Macromolecule #1: DNA polymerase alpha
Macromolecule | Name: DNA polymerase alpha / type: protein_or_peptide / ID: 1 / Name.synonym: DNA polymerase alpha / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast |
Molecular weight | Experimental: 130 KDa / Theoretical: 130 KDa |
Sequence | GO: alpha DNA polymerase:primase complex, mitochondrion, DNA binding, DNA-directed DNA polymerase activity, nucleoside binding, nucleotide binding, protein binding, DNA replication initiation, DNA ...GO: alpha DNA polymerase:primase complex, mitochondrion, DNA binding, DNA-directed DNA polymerase activity, nucleoside binding, nucleotide binding, protein binding, DNA replication initiation, DNA synthesis involved in DNA repair, lagging strand elongation, premeiotic DNA replication, RNA-templated DNA biosynthetic process InterPro: DNA-directed DNA polymerase, family B, DNA-directed DNA polymerase, family B, multifunctional domain, INTERPRO: IPR017966, DNA-directed DNA polymerase, family B, conserved site, DNA- ...InterPro: DNA-directed DNA polymerase, family B, DNA-directed DNA polymerase, family B, multifunctional domain, INTERPRO: IPR017966, DNA-directed DNA polymerase, family B, conserved site, DNA-directed DNA polymerase, family B, exonuclease domain, INTERPRO: IPR004578, Zinc finger, DNA-directed DNA polymerase, family B, alpha |
-Macromolecule #2: B subunit
Macromolecule | Name: B subunit / type: protein_or_peptide / ID: 2 / Name.synonym: B subunit / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast |
Molecular weight | Experimental: 55 KDa / Theoretical: 55 KDa |
Sequence | GO: alpha DNA polymerase:primase complex, nuclear envelope, DNA binding, DNA-directed DNA polymerase activity, protein binding, DNA replication initiation, lagging strand elongation, telomere capping InterPro: DNA polymerase alpha/delta/epsilon, subunit B, DNA polymerase alpha, subunit B, DNA polymerase alpha, subunit B, N-terminal |
-Experimental details
-Structure determination
Method | negative staining |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.7 mg/mL |
---|---|
Buffer | pH: 8 / Details: 100mM TrisHCl pH 8.0, 500 mM NaCl |
Staining | Type: NEGATIVE Details: A diluted solution of Pol alpha complex was adsorbed onto glow-discharged carbon coated grids, stained with 2% uranyl formate |
Grid | Details: 400 mesh copper grid |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | JEOL 1230 |
---|---|
Temperature | Min: 293 K / Max: 293 K / Average: 293 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification |
Details | Microscope used JEOL-1230 |
Date | Aug 20, 2009 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 170 / Average electron dose: 15 e/Å2 / Bits/pixel: 8 |
Electron beam | Acceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.9 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: Eucentric / Specimen holder model: JEOL |
-Image processing
CTF correction | Details: reverse phases |
---|---|
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, Xmipp / Number images used: 12913 |
Final two d classification | Number classes: 190 |
-Atomic model buiding 1
Initial model | PDB ID: |
---|---|
Software | Name: adpem |
Details | Protocol: Rigid Body |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation |
-Atomic model buiding 2
Initial model | PDB ID: |
---|---|
Software | Name: adpem |
Details | Protocol: Rigid Body |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation |