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- EMDB-18134: Cryo-EM structure of the DNA polymerase holoenzyme E9-A20-D4 of v... -

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Basic information

Entry
Database: EMDB / ID: EMD-18134
TitleCryo-EM structure of the DNA polymerase holoenzyme E9-A20-D4 of vaccinia virus
Map dataSharpened map with b=160 A2, masked.
Sample
  • Complex: E9-A20-D4 DNA polymerase holoenzyme
    • Protein or peptide: Uracil-DNA glycosylase
    • Protein or peptide: DNA polymerase processivity factor component OPG148
    • Protein or peptide: DNA polymerase
KeywordsDNA polymerase / holoenzyme / processivity factor / uracil-DNA glycosylase / VIRAL PROTEIN
Function / homology
Function and homology information


uracil-DNA glycosylase / viral DNA genome replication / uracil DNA N-glycosylase activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA-templated DNA replication / DNA recombination / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / hydrolase activity ...uracil-DNA glycosylase / viral DNA genome replication / uracil DNA N-glycosylase activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA-templated DNA replication / DNA recombination / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / hydrolase activity / DNA repair / nucleotide binding / DNA binding
Similarity search - Function
DNA-directed DNA polymerase, family B, viral insert domain / DNA polymerase B exonuclease, N-terminal / DNA polymerase family B viral insert / DNA polymerase family B exonuclease domain, N-terminal / Chordopoxvirus A20R / Chordopoxvirus A20R protein / Uracil-DNA glycosylase, active site / Uracil-DNA glycosylase signature. / Uracil-DNA glycosylase-like domain superfamily / : ...DNA-directed DNA polymerase, family B, viral insert domain / DNA polymerase B exonuclease, N-terminal / DNA polymerase family B viral insert / DNA polymerase family B exonuclease domain, N-terminal / Chordopoxvirus A20R / Chordopoxvirus A20R protein / Uracil-DNA glycosylase, active site / Uracil-DNA glycosylase signature. / Uracil-DNA glycosylase-like domain superfamily / : / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase / Uracil-DNA glycosylase / DNA polymerase processivity factor component OPG148
Similarity search - Component
Biological speciesVaccinia virus / Vaccinia virus Copenhagen
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsBurmeister WP / Ballandras-Colas A / Boettcher B / Grimm C
Funding support France, United States, Germany, 7 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-22-CE11-0007-01 France
Agence Nationale de la Recherche (ANR)and ANR-13-BSV8-0014 France
Agence Nationale de la Recherche (ANR)ANR-15-IDEX-0002 France
Agence Nationale de la Recherche (ANR)ANR-10-INBS-0005-02 France
Agence Nationale de la Recherche (ANR)ANR-17-EURE-0003 France
National Institutes of Health/National Center for Research Resources (NIH/NCRR)P41-GM103311 United States
German Research Foundation (DFG)Fi573/22-1 Germany
CitationJournal: PLoS Pathog / Year: 2024
Title: Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.
Authors: Wim P Burmeister / Laetitia Boutin / Aurelia C Balestra / Henri Gröger / Allison Ballandras-Colas / Stephanie Hutin / Christian Kraft / Clemens Grimm / Bettina Böttcher / Utz Fischer / ...Authors: Wim P Burmeister / Laetitia Boutin / Aurelia C Balestra / Henri Gröger / Allison Ballandras-Colas / Stephanie Hutin / Christian Kraft / Clemens Grimm / Bettina Böttcher / Utz Fischer / Nicolas Tarbouriech / Frédéric Iseni /
Abstract: The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the ...The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.
History
DepositionAug 4, 2023-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18134.png

Downloads & links

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Map

FileDownload / File: emd_18134.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map with b=160 A2, masked.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 272.384 Å		1.06 Å/pix.
x 256 pix.
= 272.384 Å		1.06 Å/pix.
x 256 pix.
= 272.384 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.224
Minimum - Maximum-1.485461 - 3.6661808
Average (Standard dev.)0.00412363 (±0.06050007)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 272.384 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: correct hand, from cryosparc NU refinement

Fileemd_18134_half_map_1.map
Annotationcorrect hand, from cryosparc NU refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: correct hand, from cryosparc NU refinement

Fileemd_18134_half_map_2.map
Annotationcorrect hand, from cryosparc NU refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E9-A20-D4 DNA polymerase holoenzyme

EntireName: E9-A20-D4 DNA polymerase holoenzyme
Components
  • Complex: E9-A20-D4 DNA polymerase holoenzyme
    • Protein or peptide: Uracil-DNA glycosylase
    • Protein or peptide: DNA polymerase processivity factor component OPG148
    • Protein or peptide: DNA polymerase

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Supramolecule #1: E9-A20-D4 DNA polymerase holoenzyme

SupramoleculeName: E9-A20-D4 DNA polymerase holoenzyme / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: heterotrimer
Source (natural)Organism: Vaccinia virus / Strain: Copenhagen
Molecular weightTheoretical: 196.825 kDa/nm

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Macromolecule #1: Uracil-DNA glycosylase

MacromoleculeName: Uracil-DNA glycosylase / type: protein_or_peptide / ID: 1 / Details: D4 carries a N-terminal Strep-tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus Copenhagen
Molecular weightTheoretical: 27.468379 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MASWSHPQFE KSGGGGGLVP RGSAMNSVTV SHAPYTITYH DDWEPVMSQL VEFYNEVASW LLRDETSPIP DKFFIQLKQP LRNKRVCVC GIDPYPKDGT GVPFESPNFT KKSIKEIASS ISRLTGVIDY KGYNLNIIDG VIPWNYYLSC KLGETKSHAI Y WDKISKLL ...String:
MASWSHPQFE KSGGGGGLVP RGSAMNSVTV SHAPYTITYH DDWEPVMSQL VEFYNEVASW LLRDETSPIP DKFFIQLKQP LRNKRVCVC GIDPYPKDGT GVPFESPNFT KKSIKEIASS ISRLTGVIDY KGYNLNIIDG VIPWNYYLSC KLGETKSHAI Y WDKISKLL LQHITKHVSV LYCLGKTDFS NIRAKLESPV TTIVGYHPAA RDRQFEKDRS FEIINVLLEL DNKAPINWAQ GF IY

UniProtKB: Uracil-DNA glycosylase

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Macromolecule #2: DNA polymerase processivity factor component OPG148

MacromoleculeName: DNA polymerase processivity factor component OPG148 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus Copenhagen
Molecular weightTheoretical: 49.247031 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MTSSADLTNL KELLSLYKSL RFSDSAAIEK YNSLVEWGTS TYWKIGVQKV ANVETSISDY YDEVKNKPFN IDPGYYIFLP VYFGSVFIY SKGKNMVELG SGNSFQIPDD MRSACNKVLD SDNGIDFLRF VLLNNRWIME DAISKYQSPV NIFKLASEYG L NIPKYLEI ...String:
MTSSADLTNL KELLSLYKSL RFSDSAAIEK YNSLVEWGTS TYWKIGVQKV ANVETSISDY YDEVKNKPFN IDPGYYIFLP VYFGSVFIY SKGKNMVELG SGNSFQIPDD MRSACNKVLD SDNGIDFLRF VLLNNRWIME DAISKYQSPV NIFKLASEYG L NIPKYLEI EIEEDTLFDD ELYSIIERSF DDKFPKISIS YIKLGELRRQ VVDFFKFSFM YIESIKVDRI GDNIFIPSVI TK SGKKILV KDVDHLIRSK VREHTFVKVK KKNTFSILYD YDGNGTETRG EVIKRIIDTI GRDYYVNGKY FSKVGSAGLK QLT NKLDIN ECATVDELVD EINKSGTVKR KIKNQSAFDL SRECLGYPEA DFITLVNNMR FKIENCKVVN FNIENTNCLN NPSI ETIYR NFNQFVSIFN VVTDVKKRLF E

UniProtKB: DNA polymerase processivity factor component OPG148

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Macromolecule #3: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus Copenhagen
Molecular weightTheoretical: 120.361484 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMDPDV RCINWFESHG ENRFLYLKSR CRNGETVFIR FPHYFYYVVT DEIYQSLSPP PFNARPLGK MRTIDIDETI SYNLDIKDRK CSVADMWLIE EPKKRSIQNA TMDEFLNISW FYISNGISPD GCYSLDEQYL T KINNGCYH ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMDPDV RCINWFESHG ENRFLYLKSR CRNGETVFIR FPHYFYYVVT DEIYQSLSPP PFNARPLGK MRTIDIDETI SYNLDIKDRK CSVADMWLIE EPKKRSIQNA TMDEFLNISW FYISNGISPD GCYSLDEQYL T KINNGCYH CDDPRNCFAK KIPRFDIPRS YLFLDIECHF DKKFPSVFIN PISHTSYCYI DLSGKRLLFT LINEEMLTEQ EI QEAVDRG CLRIQSLMEM DYERELVLCS EIVLLRIAKQ LLELTFDYVV TFNGHNFDLR YITNRLELLT GEKIIFRSPD KKE AVHLCI YERNQSSHKG VGGMANTTFH VNNNNGTIFF DLYSFIQKSE KLDSYKLDSI SKNAFSCMGK VLNRGVREMT FIGD DTTDA KGKAAAFAKV LTTGNYVTVD EDIICKVIRK DIWENGFKVV LLCPTLPNDT YKLSFGKDDV DLAQMYKDYN LNIAL DMAR YCIHDACLCQ YLWEYYGVET KTDAGASTYV LPQSMVFEYR ASTVIKGPLL KLLLETKTIL VRSETKQKFP YEGGKV FAP KQKMFSNNVL IFDYNSLYPN VCIFGNLSPE TLVGVVVSTN RLEEEINNQL LLQKYPPPRY ITVHCEPRLP NLISEIA IF DRSIEGTIPR LLRTFLAERA RYKKMLKQAT SSTEKAIYDS MQYTYKIVAN SVYGLMGFRN SALYSYASAK SCTSIGRR M ILYLESVLNG AELSNGMLRF ANPLSNPFYM DDRDINPIVK TSLPIDYRFR FRSVYGDTDS VFTEIDSQDV DKSIEIAKE LERLINNRVL FNNFKIEFEA VYKNLIMQSK KKYTTMKYSA SSNSKSVPER INKGTSETRR DVSKFHKNMI KTYKTRLSEM LSEGRMNSN QVCIDILRSL ETDLRSEFDS RSSPLELFML SRMHHSNYKS ADNPNMYLVT EYNKNNPETI ELGERYYFAY I CPANVPWT KKLVNIKTYE TIIDRSFKLG SDQRIFYEVY FKRLTSEIVN LLDNKVLCIS FFERMFGSKP TFYEA

UniProtKB: DNA polymerase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.3 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
35.0 mMC4H15Cl3NO3Tris-HCl
5.0 mMC10H16N2O8EDTA
3.8 mMC10H18N2O3desthiobiotin

Details: 35 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 3.8 mM desthiobiotin
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.013000000000000001 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1376 / Average electron dose: 68.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 42000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 381864
Startup modelType of model: NONE
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.2.1) / Software - details: NU-Refine / Number images used: 104239
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v4.2.1) / Software - details: 2D Class
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v4.2.1) / Software - details: Ab initio
Final 3D classificationNumber classes: 4 / Avg.num./class: 40000 / Software - Name: cryoSPARC (ver. v4.2.1) / Software - details: Hetero Refine
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

4od8

chain_id: D, source_name: PDB, initial_model_type: experimental modelalso A1-A46

0075

chain_id: E, source_name: Other, initial_model_type: experimental modelalso A304-A426

8hg1

chain_id: A, source_name: PDB, initial_model_type: experimental modelonly A47-A303
DetailsPhenix real-space refinement
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8q3r:
Cryo-EM structure of the DNA polymerase holoenzyme E9-A20-D4 of vaccinia virus

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