|Entry||Database: EMDB / ID: 3458|
|Title||negative-stain volume of Sso DNA PolB1|
|Map data||Final map from iteration 10 onto which FSC has been calculated and displayed (Figure S4C, right). Contour level in Chimera|
|Sample||archaeal DNA polymerase B1|
|Method||Negative stain / single particle reconstruction / 22.8 Å resolution|
|Authors||Abrescia NGA / Bell SD|
|Citation||Journal: Nat Commun / Year: 2017|
Title: Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.
Authors: Jiangyu Yan / Thomas R Beattie / Adriana L Rojas / Kelly Schermerhorn / Tamzin Gristwood / Jonathan C Trinidad / Sonja V Albers / Pietro Roversi / Andrew F Gardner / Nicola G A Abrescia / Stephen D Bell
Abstract: Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family ...Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.
|Date||Deposition: Nov 3, 2016 / Header (metadata) release: Dec 7, 2016 / Map release: May 17, 2017 / Last update: Aug 2, 2017|
Downloads & links
|File||emd_3458.map.gz (map file in CCP4 format, 8389 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 1.66 Å|
CCP4 map header:
|File||emd_3458_msk_1.map ( map file in CCP4 format, 8389 KB )|
|Projections & Slices|
|Data type||Image stored as Reals|
|Annotation details||mask created using the initial reference map (Figure S4B) with soft edges.|
|Space group number||1|
-Entire archaeal DNA polymerase B1
|Entire||Name: archaeal DNA polymerase B1 / Details: This is the apo enzyme. / Number of components: 2|
|Mass||Theoretical: 101 kDa|
-Component #1: protein, archaeal DNA polymerase B1
|Protein||Name: archaeal DNA polymerase B1 / Details: This is the apo enzyme. / Recombinant expression: No|
|Mass||Theoretical: 101 kDa|
|Source||Species: Archaea /|
|Source (engineered)||Expression System: Escherichia coli / / bacteria / / Vector: pET33b|
-Component #2: protein, DNA polymerase B1
|Protein||Name: DNA polymerase B1 / Recombinant expression: No|
|Source (engineered)||Expression System: Archaea / / Vector: pET33b|
|Specimen||Specimen state: particle / Method: Negative stain|
|Sample solution||Specimen conc.: 0.01 mg/ml / pH: 7.5|
|Staining||Negatively stained EM specimens were prepared using carbon-coated grids and stained with 2% of uranyl formate solution.|
|Vitrification||Cryogen name: NONE|
-Electron microscopy imaging
|Imaging||Microscope: JEOL 2200FSC|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 30 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 60000 X (nominal), 90201 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1300 - 1700 nm / Energy filter: In-column Omega Filter / Energy window: 0-20 eV|
|Specimen Holder||Model: JEOL|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Image acquisition||Number of digital images: 108|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 14582|
|3D reconstruction||Software: RELION|
CTF correction: phase flipping was performed using XMIPP software
Resolution: 22.8 Å / Resolution method: FSC 0.5 CUT-OFF
Details: Calculated for the converged iteration 10 in Relion using Scipion
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