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- EMDB-36962: F8-A22-E4 complex of MPXV in complex with DNA and dCTP -

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Basic information

Entry
Database: EMDB / ID: EMD-36962
TitleF8-A22-E4 complex of MPXV in complex with DNA and dCTP
Map data
Sample
  • Complex: F8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP
    • Protein or peptide: DNA polymerase F8
    • Protein or peptide: Uracil-DNA glycosylase E4
    • Protein or peptide: DNA polymerase processivity factor component A20
    • DNA: DNA (5'-D(*AP*TP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')
    • DNA: DNA (5'-D(P*AP*AP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*AP*T)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: CYTIDINE-5'-TRIPHOSPHATE
  • Ligand: water
KeywordsRECOMBINATION / REPLICATION / VIRAL PROTEIN-DNA COMPLEX
Function / homologyChordopoxvirus A20R / Chordopoxvirus A20R protein / DNA replication / DNA polymerase processivity factor
Function and homology information
Biological speciesMonkeypox virus / DNA molecule (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsShen YP / Li YN / Yan RH
Funding support China, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0509301 China
CitationJournal: Structure / Year: 2024
Title: Structural basis for the inhibition mechanism of the DNA polymerase holoenzyme from mpox virus.
Authors: Yaping Shen / Yaning Li / Renhong Yan /
Abstract: There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is ...There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is recognized as a vital antiviral target because MPXV replicates in the cytoplasm of host cells. Nucleotide analogs such as cidofovir and cytarabine (Ara-C) have shown potential in curbing MPXV replication and they also display promise against other poxviruses. However, the mechanism behind their inhibitory effects remains unclear. Here, we present the cryo-EM structure of the DNA polymerase holoenzyme F8/A22/E4 bound with its competitive inhibitor Ara-C-derived cytarabine triphosphate (Ara-CTP) at an overall resolution of 3.0 Å and reveal its inhibition mechanism. Ara-CTP functions as a direct chain terminator in proximity to the deoxycytidine triphosphate (dCTP)-binding site. The extra hydrogen bond formed with Asn665 makes it more potent in binding than dCTP. Asn665 is conserved among eukaryotic B-family polymerases.
History
DepositionJul 31, 2023-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36962.png

Downloads & links

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Map

FileDownload / File: emd_36962.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 256 pix.
= 278.272 Å		1.09 Å/pix.
x 256 pix.
= 278.272 Å		1.09 Å/pix.
x 256 pix.
= 278.272 Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.087 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-4.585059 - 6.1257057
Average (Standard dev.)0.0016824614 (±0.108753026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 278.272 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36962_half_map_1.map
Projections & Slices
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Density Histograms

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Histogram (log scale)

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Half map: #1

Fileemd_36962_half_map_2.map
Projections & Slices
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Sample components

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Entire : F8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP

EntireName: F8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP
Components
  • Complex: F8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP
    • Protein or peptide: DNA polymerase F8
    • Protein or peptide: Uracil-DNA glycosylase E4
    • Protein or peptide: DNA polymerase processivity factor component A20
    • DNA: DNA (5'-D(*AP*TP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')
    • DNA: DNA (5'-D(P*AP*AP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*AP*T)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: CYTIDINE-5'-TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: F8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP

SupramoleculeName: F8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Monkeypox virus

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Macromolecule #1: DNA polymerase F8

MacromoleculeName: DNA polymerase F8 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 117.045055 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDVRCINWFE SHGENRFLYL KSRCRNGETV FIRFPHYFYY VVTDEIYQSL SPPPFNARPM GKMRTIDIDE TISYNLDIKD RKCSVADMW LIEEPKKRSI QNATMDEFFN ISWFYISNGI SPDGCYSLDE QYLTKINNGC YHCDDPRNCF AKEIPRFDIP R SYLFLAIA ...String:
MDVRCINWFE SHGENRFLYL KSRCRNGETV FIRFPHYFYY VVTDEIYQSL SPPPFNARPM GKMRTIDIDE TISYNLDIKD RKCSVADMW LIEEPKKRSI QNATMDEFFN ISWFYISNGI SPDGCYSLDE QYLTKINNGC YHCDDPRNCF AKEIPRFDIP R SYLFLAIA 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

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Macromolecule #2: Uracil-DNA glycosylase E4

MacromoleculeName: Uracil-DNA glycosylase E4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
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

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Macromolecule #3: DNA polymerase processivity factor component A20

MacromoleculeName: DNA polymerase processivity factor component A20 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / 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 #4: DNA (5'-D(*AP*TP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')

MacromoleculeName: DNA (5'-D(*AP*TP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 3.518307 KDa
SequenceString:
(DA)(DT)(DC)(DC)(DT)(DC)(DC)(DC)(DC)(DT) (DA)(DC)

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Macromolecule #5: DNA (5'-D(P*AP*AP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*AP*AP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*AP*T)-3')
type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 5.067301 KDa
SequenceString:
(DT)(DA)(DA)(DG)(DG)(DT)(DA)(DG)(DG)(DG) (DG)(DA)(DG)(DG)(DA)(DT)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: CYTIDINE-5'-TRIPHOSPHATE

MacromoleculeName: CYTIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: CTP
Molecular weightTheoretical: 483.156 Da
Chemical component information

ChemComp-CTP:
CYTIDINE-5'-TRIPHOSPHATE

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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 (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: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 392167
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD

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