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- EMDB-3462: negative-stain 3D reconstruction of Sso heterotrimeric holo DNA-PolB1 -

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Basic information

Entry
Database: EMDB / ID: 3462
Titlenegative-stain 3D reconstruction of Sso heterotrimeric holo DNA-PolB1
Map dataFinal map for holo-PolB1* - no masked.Contoured in Chimera at 0.03 threshold. FSC have been calculated for this map at convergence.
Samplearchaeal heterotrimeric DNA polymerase B1 holo-complex
SourceArchaea /
Methodsingle particle reconstruction, at 20.2 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 9, 2016 / Header (metadata) release: Dec 7, 2016 / Map release: May 17, 2017 / Last update: Sep 20, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF CHIMERA
  • Download
3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_3462.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:0.03 (by author), 0.03 (movie #1):
Minimum - Maximum-0.047942128 - 0.09896218
Average (Standard dev.)0.0005669667 (0.008588606)
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-0.0480.0990.001

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Supplemental data

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Sample components

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Entire archaeal heterotrimeric DNA polymerase B1 holo-complex

EntireName: archaeal heterotrimeric DNA polymerase B1 holo-complex
Number of components: 5
MassTheoretical: 130 kDa

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Component #1: protein, archaeal heterotrimeric DNA polymerase B1 holo-complex

ProteinName: archaeal heterotrimeric DNA polymerase B1 holo-complex
Recombinant expression: No
MassTheoretical: 130 kDa
SourceSpecies: Archaea /
Source (engineered)Expression System: Escherichia coli / bacteria / / / Vector: pET33b/pET30a

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Component #2: protein, Sulfolobus solfataricus DNA polymerase B1 enzime holo-complex

ProteinName: Sulfolobus solfataricus DNA polymerase B1 enzime holo-complex
Details: The sample sequence contemplates the DNA polB1 but complex investigated by negative stain EM is formed by the PBP1, PBP2 and DNA. > DNA-oligo1 fragment ACAGGTAAGCAGTCCGCG > DNA-oligo2 fragment GCGGACTGCTTACDDC
Recombinant expression: No
Source (engineered)Expression System: Archaea / / Vector: pET33b

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Component #3: protein, Polymerase Binding Protein 1 - delta CTD

ProteinName: Polymerase Binding Protein 1 - delta CTD / Recombinant expression: No
Source (engineered)Expression System: Archaea / / Vector: pET30a

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Component #4: protein, Polymerase Binding Protein 2

ProteinName: Polymerase Binding Protein 2 / Recombinant expression: No
Source (engineered)Expression System: Archaea / / Vector: pET30a

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Component #5: nucleic-acid, DNA fragment

Nucleic-acidName: DNA fragment / Class: DNA / Details: oligonucleotide primer-template junction / Structure: OTHER / Synthetic: No
Sequence:
ACAGGTAAGC AGTCCGCGGC GGACTGCTTA CDDC

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Experimental details

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Sample preparation

Specimen stateparticle
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: 400

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 11758
3D reconstructionSoftware: RELION
CTF correction: phase flipping was performed using XMIPP software
Resolution: 20.2 Å / Resolution method: FSC 0.5 CUT-OFF
Details: Calculated for the converged iteration 13 in Relion using Scipion

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