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- EMDB-34886: F8-A22-E4 complex of MPXV in hexameric form -

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Basic information

Entry
Database: EMDB / ID: EMD-34886
TitleF8-A22-E4 complex of MPXV in hexameric form
Map data
Sample
  • Complex: F8-A22-E4 complex of MPXV in hexameric form
    • Complex: DNA polymerase processivity factor component A20
      • Protein or peptide: DNA polymerase processivity factor component A20
    • Complex: E4R
      • Protein or peptide: E4R
    • Complex: DNA polymerase
      • Protein or peptide: DNA polymerase
KeywordsMPXV / complex / RECOMBINATION / REPLICATION
Function / homology
Function and homology information


viral DNA genome replication / uracil DNA N-glycosylase activity / DNA-templated DNA replication / DNA recombination / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase 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 / DNA polymerase processivity factor / Uracil-DNA glycosylase
Similarity search - Component
Biological speciesMonkeypox virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLi YN / Shen YP / Hu ZW / Yan RH
Funding support China, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0509301 China
CitationJournal: Sci Adv / Year: 2023
Title: Structural basis for the assembly of the DNA polymerase holoenzyme from a monkeypox virus variant.
Authors: Yaning Li / Yaping Shen / Ziwei Hu / Renhong Yan /
Abstract: The ongoing global pandemic caused by a variant of the monkeypox (or mpox) virus (MPXV) has prompted widespread concern. The MPXV DNA polymerase holoenzyme, consisting of F8, A22, and E4, is vital ...The ongoing global pandemic caused by a variant of the monkeypox (or mpox) virus (MPXV) has prompted widespread concern. The MPXV DNA polymerase holoenzyme, consisting of F8, A22, and E4, is vital for replicating the viral genome and represents a crucial target for the development of antiviral drugs. However, the assembly and working mechanism for the DNA polymerase holoenzyme of MPXV remains elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the DNA polymerase holoenzyme at an overall resolution of 3.5 Å. Unexpectedly, the holoenzyme is assembled as a dimer of heterotrimers, of which the extra interface between the thumb domain of F8 and A22 shows a clash between A22 and substrate DNA, suggesting an autoinhibition state. Addition of exogenous double-stranded DNA shifts the hexamer into trimer exposing DNA binding sites, potentially representing a more active state. Our findings provide crucial steps toward developing targeted antiviral therapies for MPXV and related viruses.
History
DepositionDec 2, 2022-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_34886.png

Downloads & links

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Map

FileDownload / File: emd_34886.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 288 pix.
= 315.36 Å		1.1 Å/pix.
x 288 pix.
= 315.36 Å		1.1 Å/pix.
x 288 pix.
= 315.36 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.095 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.042819813 - 0.08591359
Average (Standard dev.)0.000054715343 (±0.0019838975)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 315.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34886_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34886_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : F8-A22-E4 complex of MPXV in hexameric form

EntireName: F8-A22-E4 complex of MPXV in hexameric form
Components
  • Complex: F8-A22-E4 complex of MPXV in hexameric form
    • Complex: DNA polymerase processivity factor component A20
      • Protein or peptide: DNA polymerase processivity factor component A20
    • Complex: E4R
      • Protein or peptide: E4R
    • Complex: DNA polymerase
      • Protein or peptide: DNA polymerase

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Supramolecule #1: F8-A22-E4 complex of MPXV in hexameric form

SupramoleculeName: F8-A22-E4 complex of MPXV in hexameric form / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Monkeypox virus

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Supramolecule #2: DNA polymerase processivity factor component A20

SupramoleculeName: DNA polymerase processivity factor component A20 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Monkeypox virus

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Supramolecule #3: E4R

SupramoleculeName: E4R / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Monkeypox virus

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Supramolecule #4: DNA polymerase

SupramoleculeName: DNA polymerase / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3

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Macromolecule #1: DNA polymerase processivity factor component A20

MacromoleculeName: DNA polymerase processivity factor component A20 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 49.203926 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTSSADLTNL KELLSLYKSL RFSDSVAIEK YNSLVEWGTS TYWKIGVQKV TNVETSISDY YDEVKNKPFN IDPGYYIFLP VYFGSVFIY SKGKNMVELG SGNSFQIPDE IRSACNKVLD SDNGIDFLRF VLLNNRWIME DAISKYQSPV NIFKLASEYG L NIPNYLEI ...String:
MTSSADLTNL KELLSLYKSL RFSDSVAIEK YNSLVEWGTS TYWKIGVQKV TNVETSISDY YDEVKNKPFN IDPGYYIFLP VYFGSVFIY SKGKNMVELG SGNSFQIPDE IRSACNKVLD SDNGIDFLRF VLLNNRWIME DAISKYQSPV NIFKLASEYG L NIPNYLEI EIEEDTLFDD ELYSIMERSF DDTFPKISIS YIKLGELKRQ VVDFFKFSFM YIESIKVDRI GDNIFIPSVI TK SGKKILV KDVDHLIRSK VREHTFVKVK KKNTFSILYD YDGNGTETRG EVIKRIIDTI GRDYYVNGKY FSKVGIAGLK QLT NKLDIN ECATVDELVD EINKSGTVKR KIKNQSVFDL SRECLGYPEA DFITLVNNMR FKIENCKVVN FNIENTNCLN NPSI ETIYG NFNQFVSIFN TVTDVKKRLF E

UniProtKB: DNA polymerase processivity factor

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Macromolecule #2: E4R

MacromoleculeName: E4R / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: uracil-DNA glycosylase
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 25.107742 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MNSVTISHAP YTITYHDDWE PVMSQLVEFY NEVASWLLRD ETSPIPDKFF IQLKQPLRNK RVCVCGIDPY PKDGTGVPFE SPNFTKKSI KEIASSISRL TGVIDYKGYN LNIIDGVIPW NYYLSCKLGE TKSHAIYWDK ISKLLLQHIT KHVSVLYCLG K TDFSNIRA ...String:
MNSVTISHAP YTITYHDDWE PVMSQLVEFY NEVASWLLRD ETSPIPDKFF IQLKQPLRNK RVCVCGIDPY PKDGTGVPFE SPNFTKKSI KEIASSISRL TGVIDYKGYN LNIIDGVIPW NYYLSCKLGE TKSHAIYWDK ISKLLLQHIT KHVSVLYCLG K TDFSNIRA KLESPVTTIV GYHPAARDHQ FEKDRSFEII NVLLELDNKT PINWAQGFIY

UniProtKB: Uracil-DNA glycosylase

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Macromolecule #3: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 117.147102 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDVRCINWFE SHGENRFLYL KSRCRNGETV FIRFPHYFYY VVTDEIYQSL SPPPFNARPM GKMRTIDIDE TISYNLDIKD RKCSVADMW LIEEPKKRSI QNATMDEFFN ISWFYISNGI SPDGCYSLDE QYLTKINNGC YHCDDPRNCF AKEIPRFDIP R SYLFLDIE ...String:
MDVRCINWFE SHGENRFLYL KSRCRNGETV FIRFPHYFYY VVTDEIYQSL SPPPFNARPM GKMRTIDIDE TISYNLDIKD RKCSVADMW LIEEPKKRSI QNATMDEFFN ISWFYISNGI SPDGCYSLDE QYLTKINNGC YHCDDPRNCF AKEIPRFDIP R SYLFLDIE CHFDKKFPSV FINPISHTSY CYIDLSGKRL LFTLINEEML TEQEIQEAVD RGCLRIQSLM EMDYERELVL CS EIVLLRI AKQLLELTFD YVVTFNGHNF DLRYITNRLE LLTGEKIIFR SPDKKEAVHL CIYERNQSSH KGVCGMANTT FHV NNNNGT IFFDLYSFIQ KSEKLDSYKL DSISKNAFSC MGKVLNRGVR EMTFIGDDTT DAKGKADTFA KVLTTGNYVT VDED IICKV IRKDILENGF KVVLSCPTLP NDIYKLSFGK DDIDLAQMYK DYNLNIALDM ARYCIHDACL CQYLWEYYGV ETKTD AGAA TYVLPQSMVF EYRASTIIKG PLLKLLLETK TILVRSETKQ KFPYEGGKVF APKQKMFSNN VLIFDYNSLY PNVCIF GNL SPETLVGVVV STNRLEEEIN NQLLLQKYPP PRYITVHCEP RLPNLISEIA IFDRSIEGTI PRLLRTFLAE RARYKKM LK QATSSTEKAI YDSMQYTYKI VANSVYGLMG FRNSALYSYA SAKSCTSIGR RMILYLESVL NGAELSNGML RFANTLSN P FYMDDRDINP IVKTSLPIDY RFRFRSVYGD TDSVFTEIDS QDVDKSIEIA KELERLINSR VLFNNFKIEF EAVYKNLIM QSKKKYTTMK YSASSNSKSV PERINKGTSE TRRDVSKFHK NMIKTYKTRL SEMLSEGRMN SNQVCIDILR SLETDLRSEF DSRSSPLEL FMLSRMHHSN YKSADNPNMY LVTEYNKNNP ETIELGERYY FAYICPANVP WTKKLVNIKT YETIIDRSFK L GSNQRIFY EVYFKRLTSE IVNLLDNKVL CISFFQRMFG SRPTFYEA

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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.4000000000000001 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6) / Number images used: 391085
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD

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