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- EMDB-3458: negative-stain volume of Sso DNA PolB1 -

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Basic information

Entry
Database: EMDB / ID: 3458
Titlenegative-stain volume of Sso DNA PolB1
Map dataFinal map from iteration 10 onto which FSC has been calculated and displayed (Figure S4C, right). Contour level in Chimera
Samplearchaeal DNA polymerase B1
  • DNA polymerase B1
SourceArchaea /
MethodNegative stain / single particle reconstruction / 22.8 Å resolution
AuthorsAbrescia NGA / Bell SD
CitationJournal: 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.
DateDeposition: Nov 3, 2016 / Header (metadata) release: Dec 7, 2016 / Map release: May 17, 2017 / Last update: Aug 2, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0236
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 0.0236
  • Imaged by UCSF CHIMERA
  • Download
3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_3458.map.gz (map file in CCP4 format, 8389 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
128 pix
1.66 Å/pix.
= 212.48 Å
128 pix
1.66 Å/pix.
= 212.48 Å
128 pix
1.66 Å/pix.
= 212.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.66 Å
Density
Contour Level:0.025 (by author), 0.0236 (movie #1):
Minimum - Maximum-0.04231351 - 0.08533787
Average (Standard dev.)0.0006148041 (0.006312904)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin000
Limit127127127
Spacing128128128
CellA=B=C: 212.48 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.661.661.66
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z212.480212.480212.480
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0420.0850.001

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Supplemental data

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Mask #1

Fileemd_3458_msk_1.map ( map file in CCP4 format, 8389 KB )
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms
Data typeImage stored as Reals
Annotation detailsmask created using the initial reference map (Figure S4B) with soft edges.
Space group number1

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Mask #1~

Fileemd_3458_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire archaeal DNA polymerase B1

EntireName: archaeal DNA polymerase B1 / Details: This is the apo enzyme. / Number of components: 2
MassTheoretical: 101 kDa

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Component #1: protein, archaeal DNA polymerase B1

ProteinName: archaeal DNA polymerase B1 / Details: This is the apo enzyme. / Recombinant expression: No
MassTheoretical: 101 kDa
SourceSpecies: Archaea /
Source (engineered)Expression System: Escherichia coli / / bacteria / / Vector: pET33b

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Component #2: protein, DNA polymerase B1

ProteinName: DNA polymerase B1 / Recombinant expression: No
Source (engineered)Expression System: Archaea / / Vector: pET33b

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: Negative stain
Sample solutionSpecimen conc.: 0.01 mg/ml / pH: 7.5
StainingNegatively stained EM specimens were prepared using carbon-coated grids and stained with 2% of uranyl formate solution.
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 2200FSC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 30 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 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 HolderModel: JEOL
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 108

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 14582
3D reconstructionSoftware: 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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