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- EMDB-36068: Monkeypox virus DNA replication holoenzyme F8, A22 and E4 in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-36068
TitleMonkeypox virus DNA replication holoenzyme F8, A22 and E4 in complex with a DNA duplex and dCTP
Map data
Sample
  • Complex: Monkeypox virus replication holoenzyme F8-A22-E4 in complex with a DNA duplex and dCTP
    • Protein or peptide: DNA polymerase
    • Protein or peptide: E4R
    • Protein or peptide: DNA polymerase processivity factor component A20
    • DNA: DNA (5'-D(P*AP*GP*CP*TP*GP*CP*TP*AP*TP*GP*AP*GP*AP*TP*TP*AP*AP*GP*TP*TP*AP*T)-3')
    • DNA: DNA (5'-D(P*GP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*GP*AP*TP*AP*AP*CP*TP*TP*AP*AP*TP*CP*TP*CP*AP*CP*AP*TP*AP*GP*CP*AP*GP*CP*T)-3')
  • Ligand: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM ION
KeywordsMonkeypox virus / DNA replication holoenzyme / DNA replication machinery / DNA polymerase / B-family DNA polymerase / uracil-DNA glycosylase / MPXV / orthopoxvirus / poxviridae / DNA processivity factor / VIRAL PROTEIN
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 processivity factor / Uracil-DNA glycosylase / DNA polymerase
Similarity search - Component
Biological speciesMonkeypox virus / DNA molecule (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsXu Y / Wu Y / Wu X / Zhang Y / Yang Y / Li D / Yang B / Gao K / Zhang Z / Dong C ...Xu Y / Wu Y / Wu X / Zhang Y / Yang Y / Li D / Yang B / Gao K / Zhang Z / Dong C / Tang X / Dong H
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32250710142 China
CitationJournal: Int J Biol Macromol / Year: 2024
Title: Structural basis of human mpox viral DNA replication inhibition by brincidofovir and cidofovir.
Authors: Yunxia Xu / Yaqi Wu / Xiaoying Wu / Yuanyuan Zhang / Yaxue Yang / Danyang Li / Biao Yang / Kaiting Gao / Zhengyu Zhang / Changjiang Dong /
Abstract: Mpox virus has wildly spread over 108 non-endemic regions in the world since May 2022. DNA replication of mpox is performed by DNA polymerase machinery F8-A22-E4, which is known as a great drug ...Mpox virus has wildly spread over 108 non-endemic regions in the world since May 2022. DNA replication of mpox is performed by DNA polymerase machinery F8-A22-E4, which is known as a great drug target. Brincidofovir and cidofovir are reported to have broad-spectrum antiviral activity against poxviruses, including mpox virus in animal models. However, the molecular mechanism is not understood. Here we report cryogenic electron microscopy structures of mpox viral F8-A22-E4 in complex with a DNA duplex, or dCTP and the DNA duplex, or cidofovir diphosphate and the DNA duplex at resolution of 3.22, 2.98 and 2.79 Å, respectively. Our structural work and DNA replication inhibition assays reveal that cidofovir diphosphate is located at the dCTP binding position with a different conformation to compete with dCTP to incorporate into the DNA and inhibit DNA synthesis. Conformation of both F8-A22-E4 and DNA is changed from the pre-dNTP binding state to DNA synthesizing state after dCTP or cidofovir diphosphate is bound, suggesting a coupling mechanism. This work provides the structural basis of DNA synthesis inhibition by brincidofovir and cidofovir, providing a rational strategy for new therapeutical development for mpox virus and other pox viruses.
History
DepositionMay 1, 2023-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36068.png

Downloads & links

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Map

FileDownload / File: emd_36068.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 400 pix.
= 336. Å		0.84 Å/pix.
x 400 pix.
= 336. Å		0.84 Å/pix.
x 400 pix.
= 336. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-1.3049023 - 1.8077496
Average (Standard dev.)-0.0002332232 (±0.026850484)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36068_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_36068_half_map_2.map
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Sample components

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Entire : Monkeypox virus replication holoenzyme F8-A22-E4 in complex with ...

EntireName: Monkeypox virus replication holoenzyme F8-A22-E4 in complex with a DNA duplex and dCTP
Components
  • Complex: Monkeypox virus replication holoenzyme F8-A22-E4 in complex with a DNA duplex and dCTP
    • Protein or peptide: DNA polymerase
    • Protein or peptide: E4R
    • Protein or peptide: DNA polymerase processivity factor component A20
    • DNA: DNA (5'-D(P*AP*GP*CP*TP*GP*CP*TP*AP*TP*GP*AP*GP*AP*TP*TP*AP*AP*GP*TP*TP*AP*T)-3')
    • DNA: DNA (5'-D(P*GP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*GP*AP*TP*AP*AP*CP*TP*TP*AP*AP*TP*CP*TP*CP*AP*CP*AP*TP*AP*GP*CP*AP*GP*CP*T)-3')
  • Ligand: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM ION

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Supramolecule #1: Monkeypox virus replication holoenzyme F8-A22-E4 in complex with ...

SupramoleculeName: Monkeypox virus replication holoenzyme F8-A22-E4 in complex with a DNA duplex and dCTP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 / Details: F8-A22-E4 in complex with a DNA duplex and dCTP
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 191 kDa/nm

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Macromolecule #1: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 120.041156 KDa
Recombinant expressionOrganism: Insect expression vector pBlueBachsGCA1 (others)
SequenceString: MHHHHHHHHD YDIPTTENLY FQGMDVRCIN WFESHGENRF LYLKSRCRNG ETVFIRFPHY FYYVVTDEIY QSLSPPPFNA RPMGKMRTI DIDETISYNL DIKDRKCSVA DMWLIEEPKK RSIQNATMDE FFNISWFYIS NGISPDGCYS LDEQYLTKIN N GCYHCDDP ...String:
MHHHHHHHHD YDIPTTENLY FQGMDVRCIN WFESHGENRF LYLKSRCRNG ETVFIRFPHY FYYVVTDEIY QSLSPPPFNA RPMGKMRTI DIDETISYNL DIKDRKCSVA DMWLIEEPKK RSIQNATMDE FFNISWFYIS NGISPDGCYS LDEQYLTKIN N GCYHCDDP RNCFAKEIPR FDIPRSYLFL DIECHFDKKF PSVFINPISH TSYCYIDLSG KRLLFTLINE EMLTEQEIQE AV DRGCLRI QSLMEMDYER ELVLCSEIVL LRIAKQLLEL TFDYVVTFNG HNFDLRYITN RLELLTGEKI IFRSPDKKEA VHL CIYERN QSSHKGVCGM ANTTFHVNNN NGTIFFDLYS FIQKSEKLDS YKLDSISKNA FSCMGKVLNR GVREMTFIGD DTTD AKGKA DTFAKVLTTG NYVTVDEDII CKVIRKDILE NGFKVVLSCP TLPNDIYKLS FGKDDIDLAQ MYKDYNLNIA LDMAR YCIH DACLCQYLWE YYGVETKTDA GAATYVLPQS MVFEYRASTI IKGPLLKLLL ETKTILVRSE TKQKFPYEGG KVFAPK QKM FSNNVLIFDY NSLYPNVCIF GNLSPETLVG VVVSTNRLEE EINNQLLLQK YPPPRYITVH CEPRLPNLIS EIAIFDR SI EGTIPRLLRT FLAERARYKK MLKQATSSTE KAIYDSMQYT YKIVANSVYG LMGFRNSALY SYASAKSCTS IGRRMILY L ESVLNGAELS NGMLRFANTL SNPFYMDDRD INPIVKTSLP IDYRFRFRSV YGDTDSVFTE IDSQDVDKSI EIAKELERL INSRVLFNNF KIEFEAVYKN LIMQSKKKYT TMKYSASSNS KSVPERINKG TSETRRDVSK FHKNMIKTYK TRLSEMLSEG RMNSNQVCI DILRSLETDL RSEFDSRSSP LELFMLSRMH HSNYKSADNP NMYLVTEYNK NNPETIELGE RYYFAYICPA N VPWTKKLV NIKTYETIID RSFKLGSNQR IFYEVYFKRL TSEIVNLLDN KVLCISFFQR MFGSRPTFYE A

UniProtKB: DNA polymerase

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Macromolecule #2: E4R

MacromoleculeName: E4R / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: uracil-DNA glycosylase
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 27.883709 KDa
Recombinant expressionOrganism: Insect expression vector pBlueBachsGCA1 (others)
SequenceString: MHHHHHHDYD IPTTENLYFQ GASMNSVTIS HAPYTITYHD DWEPVMSQLV EFYNEVASWL LRDETSPIPD KFFIQLKQPL RNKRVCVCG IDPYPKDGTG VPFESPNFTK KSIKEIASSI SRLTGVIDYK GYNLNIIDGV IPWNYYLSCK LGETKSHAIY W DKISKLLL ...String:
MHHHHHHDYD IPTTENLYFQ GASMNSVTIS HAPYTITYHD DWEPVMSQLV EFYNEVASWL LRDETSPIPD KFFIQLKQPL RNKRVCVCG IDPYPKDGTG VPFESPNFTK KSIKEIASSI SRLTGVIDYK GYNLNIIDGV IPWNYYLSCK LGETKSHAIY W DKISKLLL QHITKHVSVL YCLGKTDFSN IRAKLESPVT TIVGYHPAAR DHQFEKDRSF EIINVLLELD NKTPINWAQG FI Y

UniProtKB: Uracil-DNA glycosylase

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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: Insect expression vector pBlueBachsGCA1 (others)
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(P*AP*GP*CP*TP*GP*CP*TP*AP*TP*GP*AP*GP*AP*TP*TP*AP*AP*GP...

MacromoleculeName: DNA (5'-D(P*AP*GP*CP*TP*GP*CP*TP*AP*TP*GP*AP*GP*AP*TP*TP*AP*AP*GP*TP*TP*AP*T)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 7.438825 KDa
SequenceString:
(DA)(DG)(DC)(DT)(DG)(DC)(DT)(DA)(DT)(DG) (DT)(DG)(DA)(DG)(DA)(DT)(DT)(DA)(DA)(DG) (DT)(DT)(DA)(DT)

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Macromolecule #5: DNA (5'-D(P*GP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*GP*AP*TP*AP*AP...

MacromoleculeName: DNA (5'-D(P*GP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*GP*AP*TP*AP*AP*CP*TP*TP*AP*AP*TP*CP*TP*CP*AP*CP*AP*TP*AP*GP*CP*AP*GP*CP*T)-3')
type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 11.60548 KDa
SequenceString:
(DG)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DG)(DA)(DT)(DA)(DA)(DC)(DT) (DT)(DA)(DA)(DT)(DC)(DT)(DC)(DA)(DC) (DA)(DT)(DA)(DG)(DC)(DA)(DG)(DC)(DT)

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Macromolecule #6: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: DCP
Molecular weightTheoretical: 467.157 Da
Chemical component information

ChemComp-DCP:
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

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Macromolecule #7: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 7 / Number of copies: 3 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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
Details: 25 mM MOPS, pH 7.0, 200 mM NaCl, 5% (w/v) glycerol, 1 mM TCEP
VitrificationCryogen name: ETHANE
DetailsF8-A22-E4 in complex with a DNA duplex and dCTP

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Electron microscopy

MicroscopeTFS 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: 3.0 µm / Nominal defocus min: 1.0 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

DetailsThe selected movies are corrected with motion correct
Particle selectionDetails: 15,938,666
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8j86


Details: F8-A22_E4 in complex with a DNA duplex
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 1057305
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8j8f:
Monkeypox virus DNA replication holoenzyme F8, A22 and E4 in complex with a DNA duplex and dCTP

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