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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: EMD-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.
Sample
  • Complex: archaeal heterotrimeric DNA polymerase B1 holo-complex
    • Protein or peptide: Sulfolobus solfataricus DNA polymerase B1 enzime holo-complex
    • Protein or peptide: Polymerase Binding Protein 1 - delta CTD
    • Protein or peptide: Polymerase Binding Protein 2
    • DNA: DNA fragment
Biological speciesArchaea (unknown)
Methodsingle particle reconstruction / negative staining / Resolution: 20.2 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 9, 2016-
Header (metadata) releaseDec 7, 2016-
Map releaseMay 17, 2017-
UpdateSep 20, 2017-
Current statusSep 20, 2017Processing site: PDBe / Status: Released

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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
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3462.png

Downloads & links

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Map

FileDownload / File: emd_3462.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map for holo-PolB1* - no masked.Contoured in Chimera at 0.03 threshold. FSC have been calculated for this map at convergence.
Voxel sizeX=Y=Z: 1.66 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.047942128 - 0.09896218
Average (Standard dev.)0.0005669667 (±0.008588606)
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-0.0480.0990.001

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Supplemental data

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Half map: half1 map for holo-PolB1*. Contoured in Chimera at 0.028 threshold

Fileemd_3462_half_map_1.map
Annotationhalf1 map for holo-PolB1*. Contoured in Chimera at 0.028 threshold
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half2 map for holo-PolB1*. Contoured in Chimera at 0.028 threshold

Fileemd_3462_half_map_2.map
Annotationhalf2 map for holo-PolB1*. Contoured in Chimera at 0.028 threshold
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 heterotrimeric DNA polymerase B1 holo-complex

EntireName: archaeal heterotrimeric DNA polymerase B1 holo-complex
Components
  • Complex: archaeal heterotrimeric DNA polymerase B1 holo-complex
    • Protein or peptide: Sulfolobus solfataricus DNA polymerase B1 enzime holo-complex
    • Protein or peptide: Polymerase Binding Protein 1 - delta CTD
    • Protein or peptide: Polymerase Binding Protein 2
    • DNA: DNA fragment

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Supramolecule #1: archaeal heterotrimeric DNA polymerase B1 holo-complex

SupramoleculeName: archaeal heterotrimeric DNA polymerase B1 holo-complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Archaea (unknown)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET33b/pET30a
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Sulfolobus solfataricus DNA polymerase B1 enzime holo-complex

MacromoleculeName: Sulfolobus solfataricus DNA polymerase B1 enzime holo-complex
type: protein_or_peptide / ID: 1
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
Enantiomer: LEVO
Source (natural)Organism: Archaea (unknown)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAKQLTLFD IPSSKPAKSE QNTQQSQQSA PVEEKKVVRR EWLEEAQENK IYFLLQVDYD GKKGKAVCKL FDKETQKIYA LYDNTGHKPY FLVDLEPDKV GKIPKIVRDP SFDHIETVSK IDPYTWNKFK LTKIVVRDPL AVRRLRNDVP KAYEAHIKYF NNYMYDIGLI ...String:
MAKQLTLFD IPSSKPAKSE QNTQQSQQSA PVEEKKVVRR EWLEEAQENK IYFLLQVDYD GKKGKAVCKL FDKETQKIYA LYDNTGHKPY FLVDLEPDKV GKIPKIVRDP SFDHIETVSK IDPYTWNKFK LTKIVVRDPL AVRRLRNDVP KAYEAHIKYF NNYMYDIGLI PGMPYVVKNG KLESVYLSLD EKDVEEIKKA FADSDEMTRQ MAVDWLPIFE TEIPKIKRVA IDIEVYTPVK GRIPDSQKAE FPIISIALAG SDGLKKVLVL NRNDVNEGSV KLDGISVERF NTEYELLGRF FDILLEYPIV LTFNGDDFDL PYIYFRALKL GYFPEEIPID VAGKDEAKYL AGLHIDLYKF FFNKAVRNYA FEGKYNEYNL DAVAKALLGT SKVKVDTLIS FLDVEKLIEY NFRDAEITLQ LTTFNNDLTM KLIVLFSRIS RLGIEELTRT EISTWVKNLY YWEHRKRNWL IPLKEEILAK SSNIRTSALI KGKGYKGAVV IDPPAGIFFN ITVLDFASLY PSIIRTWNLS YETVDIQQCK KPYEVKDETG EVLHIVCMDR PGITAVITGL LRDFRVKIYK KKAKNPNNSE EQKLLYDVVQ RAMKVFINAT YGVFGAETFP LYAPAVAESV TALGRYVITS TVKKAREEGL TVLYGDTDSL FLLNPPKNSL ENIIKWVKTT FNLDLEVDKT YKFVAFSGLK KNYFGVYQDG KVDIKGMLVK KRNTPEFVKK VFNEVKELMI SINSPNDVKE IKRKIVDVVK GSYEKLKNKG YNLDELAFKV MLSKPLDAYK KNTPQHVKAA LQLRPFGVNV LPRDIIYYVK VRSKDGVKPV QLAKVTEIDA EKYLEALRST FEQILRAFGV SWDEIAATMS IDSFFSYPSK GNS

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Macromolecule #2: Polymerase Binding Protein 1 - delta CTD

MacromoleculeName: Polymerase Binding Protein 1 - delta CTD / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Archaea (unknown)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ISHMSTRWLP KWKAIEIDYN NKKVTVCYDE VTRLYVCPIC SPNCAKGVST DYSTYFFNLE DLKRHLDAHK YGLWLQKKTR T

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Macromolecule #3: Polymerase Binding Protein 2

MacromoleculeName: Polymerase Binding Protein 2 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Archaea (unknown)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSVNQKEIEI AIEYFKNYIS VGEIVATMDL KARGISNPQA VISKLIEMGI IEKGEGCYNL VRKSTDKKle hhhhhh

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Macromolecule #4: DNA fragment

MacromoleculeName: DNA fragment / type: dna / ID: 4 / Details: oligonucleotide primer-template junction / Classification: DNA
Source (natural)Organism: Archaea (unknown)
SequenceString:
ACAGGTAAGC AGTCCGCG G CGGACTGCTT ACDDC

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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: 400 / Average exposure time: 0.5 sec. / Average electron dose: 30.0 e/Å2

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Image processing

Particle selectionNumber selected: 110000
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: 20.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION / Software - details: RELION through SCIPION
Details: Calculated for the converged iteration 13 in Relion using Scipion
Number images used: 11758

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Atomic model buiding 1

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

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