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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
Samplearchaeal heterotrimeric DNA polymerase B1 holo-complex
SourceArchaea
Map dataFinal map for holo-PolB1* - no masked.Contoured in Chimera at 0.03 threshold. FSC have been calculated for this map at convergence.
Methodsingle particle reconstruction, at 20.2 Å resolution
AuthorsAbrescia NGA / Bell SD
CitationNat Commun, 2017, 8, 15075-15075

Nat Commun, 2017, 8, 15075-15075 Yorodumi Papers
Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.
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

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
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3D viewer


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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
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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 / エシェリキア・コリ, 大腸菌 /
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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