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- EMDB-37714: Structure of monkeypox virus polymerase complex F8-A22-E4-H5 (tag... -

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Basic information

Entry
Database: EMDB / ID: EMD-37714
TitleStructure of monkeypox virus polymerase complex F8-A22-E4-H5 (tag-free A22) with exogenous DNA
Map data
Sample
  • Complex: F8-A22-E4-H5 (tag-free A22) with exogenous DNA
    • Complex: F8-A22-E4-H5
      • Protein or peptide: DNA polymerase
      • Protein or peptide: A22R DNA polymerase processivity factor
      • Protein or peptide: E4R Uracil-DNA glycosylase, DNA polymerase processivity factor
      • Protein or peptide: H5R late gene transcription factor
    • Complex: DNA
      • DNA: Primer DNA
      • DNA: Template DNA
  • Ligand: 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsViral DNA replication / monkeypox virus / polymerase / H5 / VIRAL PROTEIN
Biological speciesMonkeypox virus / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsWang X / Li N / Gao N
Funding support China, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, China) China
CitationJournal: Mol Cell / Year: 2023
Title: Structural insights into the assembly and mechanism of mpox virus DNA polymerase complex F8-A22-E4-H5.
Authors: Xiaohan Wang / Liangwen Ma / Ningning Li / Ning Gao /
Abstract: The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. ...The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. However, the molecular roles of these viral factors remain unclear. Here, we characterize the structures of F8-A22-E4 and F8-A22-E4-H5 complexes in the presence of different primer-template DNA substrates. E4 is located upstream of F8 on the template single-stranded DNA (ssDNA) and is catalytically active, highlighting a functional coupling between DNA base-excision repair and DNA synthesis. Moreover, H5, in the form of tetramer, binds to the double-stranded DNA (dsDNA) region downstream of F8 in a similar position as PCNA (proliferating cell nuclear antigen) does in eukaryotic polymerase complexes. Omission of H5 or disruption of its DNA interaction showed a reduced synthesis of full-length DNA products. These structures provide snapshots for the working cycle of the polymerase and generate insights into the mechanisms of these essential factors in viral DNA replication.
History
DepositionOct 10, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37714.png

Downloads & links

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Map

FileDownload / File: emd_37714.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.016
Minimum - Maximum-0.12648426 - 0.19866881
Average (Standard dev.)0.00019277929 (±0.003675126)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_37714_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_37714_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_37714_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : F8-A22-E4-H5 (tag-free A22) with exogenous DNA

EntireName: F8-A22-E4-H5 (tag-free A22) with exogenous DNA
Components
  • Complex: F8-A22-E4-H5 (tag-free A22) with exogenous DNA
    • Complex: F8-A22-E4-H5
      • Protein or peptide: DNA polymerase
      • Protein or peptide: A22R DNA polymerase processivity factor
      • Protein or peptide: E4R Uracil-DNA glycosylase, DNA polymerase processivity factor
      • Protein or peptide: H5R late gene transcription factor
    • Complex: DNA
      • DNA: Primer DNA
      • DNA: Template DNA
  • Ligand: 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: F8-A22-E4-H5 (tag-free A22) with exogenous DNA

SupramoleculeName: F8-A22-E4-H5 (tag-free A22) with exogenous DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Monkeypox virus

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Supramolecule #2: F8-A22-E4-H5

SupramoleculeName: F8-A22-E4-H5 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5-#6

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Macromolecule #1: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 1 / Details: GenBank: URK20494.1 / Number of copies: 1 / Enantiomer: LEVO
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

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Macromolecule #2: A22R DNA polymerase processivity factor

MacromoleculeName: A22R DNA polymerase processivity factor / type: protein_or_peptide / ID: 2 / Details: GenBank: URK20570.1 / 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

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Macromolecule #3: E4R Uracil-DNA glycosylase, DNA polymerase processivity factor

MacromoleculeName: E4R Uracil-DNA glycosylase, DNA polymerase processivity factor
type: protein_or_peptide / ID: 3 / Details: GenBank: URK20538.1 / 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 #4: H5R late gene transcription factor

MacromoleculeName: H5R late gene transcription factor / type: protein_or_peptide / ID: 4 / Details: GenBank: URK20532.1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 23.107074 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAWSITNKAD TSSFTKMAEI RAHLRNSAEN KDKNEDIFPE DVIIPSTKPK TKRTTTPRKP AATKRSTKKD KEKEEVEEVV IEEYHQTTE ENSPPPSSSP GVGDIVESVA AVELDDSDGD DEPMVQVEAG KVNHSARSDL SDLKVATDNI VKDLKKIITR I SAVSTVLE ...String:
MAWSITNKAD TSSFTKMAEI RAHLRNSAEN KDKNEDIFPE DVIIPSTKPK TKRTTTPRKP AATKRSTKKD KEKEEVEEVV IEEYHQTTE ENSPPPSSSP GVGDIVESVA AVELDDSDGD DEPMVQVEAG KVNHSARSDL SDLKVATDNI VKDLKKIITR I SAVSTVLE DVQAAGISRQ FTSMTKAITT LSDLVTEGKS KVVRKKVKTC KK

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Macromolecule #5: Primer DNA

MacromoleculeName: Primer DNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.802956 KDa
SequenceString:
(DA)(DT)(DT)(DT)(DC)(DG)(DC)(DG)(DG)(DG) (DA)(DG)(DC)(DT)(DA)(DT)(DG)(DA)(DC)(DC) (DA)(DT)(DG)(DA)(DT)(DT)(DA)(DC)(DG) (DA)(DA)(DT)(DT)(DG)(DC)

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Macromolecule #6: Template DNA

MacromoleculeName: Template DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.73649 KDa
SequenceString:
(DC)(DT)(DG)(DC)(DA)(DC)(DG)(DA)(DA)(DT) (DT)(DA)(DA)(DG)(DC)(DA)(DA)(DT)(DT)(DC) (DG)(DT)(DA)(DA)(DT)(DC)(DA)(DT)(DG) (DG)(DT)(DC)(DA)(DT)(DA)(DG)(DC)(DT)(DC) (DC) (DC)(DG)(DC)(DG)(DA)(DA)(DA)(DT)

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Macromolecule #7: 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: D3T
Molecular weightTheoretical: 466.169 Da
Chemical component information

ChemComp-23T:
2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 59.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1660000

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