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

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Basic information

Entry
Database: EMDB / ID: EMD-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
Sample
  • Complex: archaeal DNA polymerase B1
    • Protein or peptide: DNA polymerase B1
Biological speciesArchaea (unknown)
Methodsingle particle reconstruction / negative staining / Resolution: 22.8 Å
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.
History
DepositionNov 3, 2016-
Header (metadata) releaseDec 7, 2016-
Map releaseMay 17, 2017-
UpdateAug 2, 2017-
Current statusAug 2, 2017Processing site: PDBe / Status: Released

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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
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3458.png

Downloads & links

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Map

FileDownload / File: emd_3458.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map from iteration 10 onto which FSC has been calculated and displayed (Figure S4C, right). Contour level in Chimera
Voxel sizeX=Y=Z: 1.66 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.0236
Minimum - Maximum-0.04231351 - 0.08533787
Average (Standard dev.)0.0006148041 (±0.006312904)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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
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
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: This is the masked map used in Figure...

Fileemd_3458_additional.map
AnnotationThis is the masked map used in Figure 4A and in submitted figure. Contour level in Chimera.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half2 map from iteration 10. Contour level in Chimera

Fileemd_3458_half_map_1.map
Annotationhalf2 map from iteration 10. Contour level in Chimera
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half1 map from iteration 10. Contour level in Chimera

Fileemd_3458_half_map_2.map
Annotationhalf1 map from iteration 10. Contour level in Chimera
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : archaeal DNA polymerase B1

EntireName: archaeal DNA polymerase B1
Components
  • Complex: archaeal DNA polymerase B1
    • Protein or peptide: DNA polymerase B1

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Supramolecule #1: archaeal DNA polymerase B1

SupramoleculeName: archaeal DNA polymerase B1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: This is the apo enzyme.
Source (natural)Organism: Archaea (unknown)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET33b
Molecular weightTheoretical: 101 KDa

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Macromolecule #1: DNA polymerase B1

MacromoleculeName: DNA polymerase B1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Archaea (unknown)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAKQLTLFDI PSSKPAKSEQ NTQQSQQSAP VEEKKVVRRE WLEEAQENKI YFLLQVDYDG KKGKAVCKLF DKETQKIYAL YDNTGHKPYF LVDLEPDKVG KIPKIVRDPS FDHIETVSKI DPYTWNKFKL TKIVVRDPLA VRRLRNDVPK AYEAHIKYFN NYMYDIGLIP ...String:
MAKQLTLFDI PSSKPAKSEQ NTQQSQQSAP VEEKKVVRRE WLEEAQENKI YFLLQVDYDG KKGKAVCKLF DKETQKIYAL YDNTGHKPYF LVDLEPDKVG KIPKIVRDPS FDHIETVSKI DPYTWNKFKL TKIVVRDPLA VRRLRNDVPK AYEAHIKYFN NYMYDIGLIP GMPYVVKNGK LESVYLSLDE KDVEEIKKAF ADSDEMTRQM AVDWLPIFET EIPKIKRVAI DIEVYTPVKG RIPDSQKAEF PIISIALAGS DGLKKVLVLN RNDVNEGSVK LDGISVERFN TEYELLGRFF DILLEYPIVL TFNGDDFDLP YIYFRALKLG YFPEEIPIDV AGKDEAKYLA GLHIDLYKFF FNKAVRNYAF EGKYNEYNLD AVAKALLGTS KVKVDTLISF LDVEKLIEYN FRDAEITLQL TTFNNDLTMK LIVLFSRISR LGIEELTRTE ISTWVKNLYY WEHRKRNWLI PLKEEILAKS SNIRTSALIK GKGYKGAVVI DPPAGIFFNI TVLDFASLYP SIIRTWNLSY ETVDIQQCKK PYEVKDETGE VLHIVCMDRP GITAVITGLL RDFRVKIYKK KAKNPNNSEE QKLLYDVVQR AMKVFINATY GVFGAETFPL YAPAVAESVT ALGRYVITST VKKAREEGLT VLYGDTDSLF LLNPPKNSLE NIIKWVKTTF NLDLEVDKTY KFVAFSGLKK NYFGVYQDGK VDIKGMLVKK RNTPEFVKKV FNEVKELMIS INSPNDVKEI KRKIVDVVKG SYEKLKNKGY NLDELAFKVM LSKPLDAYKK NTPQHVKAAL QLRPFGVNVL PRDIIYYVKV RSKDGVKPVQ LAKVTEIDAE KYLEALRSTF EQILRAFGVS WDEIAATMSI DSFFSYPSKG NS

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMHepesHepes
100.0 mMNaClSodium chloridesodium chloride
1.0 mMDTTDithiothreitol
StainingType: NEGATIVE / Material: uranyl formate
Details: Negatively stained EM specimens were prepared using carbon-coated grids and stained with 2% of uranyl formate solution.
GridModel: EMS / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa

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Electron microscopy

MicroscopeJEOL 2200FSC
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 90201 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: JEOL
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 108 / Average exposure time: 0.5 sec. / Average electron dose: 30.0 e/Å2

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Image processing

Particle selectionNumber selected: 20000
CTF correctionSoftware - Name: CTFFIND (ver. 3) / Details: phase flipping was performed using XMIPP software
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1s5j


Details: The initial reference map was generated using the atomic model.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 3.75 degrees
Software - Name: RELION / Software - details: RELION through SCIPION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 22.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION / Software - details: RELION through SCIPION
Details: Calculated for the converged iteration 10 in Relion using Scipion
Number images used: 14582

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient

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