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- EMDB-19838: HSV-1 DNA polymerase-processivity factor complex in exonuclease s... -

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Basic information

Entry
Database: EMDB / ID: EMD-19838
TitleHSV-1 DNA polymerase-processivity factor complex in exonuclease state active site with 1-bp DNA mismatch
Map dataLocally filtered local refinement map from cryoSPARC
Sample
  • Complex: HSV-1 DNA polymerase-processivity factor complex in exonuclease state with 1-bp DNA mismatch
    • Complex: DNA polymerase-processivity factor
      • Protein or peptide: DNA polymerase catalytic subunit
    • Complex: DNA
      • DNA: DNA (46-MER)
      • DNA: DNA (67-MER)
  • Ligand: CALCIUM ION
  • Ligand: water
KeywordsDNA / Polymerase / Complex / TRANSFERASE
Function / homology
Function and homology information


DNA polymerase activity / DNA polymerase complex / bidirectional double-stranded viral DNA replication / 5'-3' exonuclease activity / ribonuclease H / DNA-templated DNA replication / RNA-DNA hybrid ribonuclease activity / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding ...DNA polymerase activity / DNA polymerase complex / bidirectional double-stranded viral DNA replication / 5'-3' exonuclease activity / ribonuclease H / DNA-templated DNA replication / RNA-DNA hybrid ribonuclease activity / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / host cell nucleus / DNA binding
Similarity search - Function
DNA polymerase catalytic subunit Pol, C-terminal / DNA polymerase catalytic subunit Pol / : / DNA polymerase family B, thumb domain / 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 catalytic subunit Pol, C-terminal / DNA polymerase catalytic subunit Pol / : / DNA polymerase family B, thumb domain / 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 catalytic subunit
Similarity search - Component
Biological speciesHuman alphaherpesvirus 1 strain KOS / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.12 Å
AuthorsGustavsson E / Grunewald K / Elias P / Hallberg BM
Funding support Sweden, Germany, 2 items
OrganizationGrant numberCountry
Swedish Research Council2017-06702 Sweden
German Research Foundation (DFG)152/772-1|152/774-1|152/775-1|152/776-1|152/777-1 FUGG Germany
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Dynamics of the Herpes simplex virus DNA polymerase holoenzyme during DNA synthesis and proof-reading revealed by Cryo-EM.
Authors: Emil Gustavsson / Kay Grünewald / Per Elias / B Martin Hällberg /
Abstract: Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, replicates using seven essential proteins encoded by its genome. Among these, the UL30 DNA polymerase, complexed with the UL42 ...Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, replicates using seven essential proteins encoded by its genome. Among these, the UL30 DNA polymerase, complexed with the UL42 processivity factor, orchestrates leading and lagging strand replication of the 152 kb viral genome. UL30 polymerase is a prime target for antiviral therapy, and resistance to current drugs can arise in immunocompromised individuals. Using electron cryo-microscopy (cryo-EM), we unveil the dynamic changes of the UL30/UL42 complex with DNA in three distinct states. First, a pre-translocation state with an open fingers domain ready for nucleotide incorporation. Second, a halted elongation state where the fingers close, trapping dATP in the dNTP pocket. Third, a DNA-editing state involving significant conformational changes to allow DNA realignment for exonuclease activity. Additionally, the flexible UL30 C-terminal domain interacts with UL42, forming an extended positively charged surface binding to DNA, thereby enhancing processive synthesis. These findings highlight substantial structural shifts in the polymerase and its DNA interactions during replication, offering insights for future antiviral drug development.
History
DepositionMar 13, 2024-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19838.png

Downloads & links

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Map

FileDownload / File: emd_19838.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally filtered local refinement map from cryoSPARC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.8 Å/pix.
x 480 pix.
= 384. Å		0.8 Å/pix.
x 480 pix.
= 384. Å		0.8 Å/pix.
x 480 pix.
= 384. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-2.0288124 - 3.128717
Average (Standard dev.)0.00029856624 (±0.022634182)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 384.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19838_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local refinement map from cryoSPARC

Fileemd_19838_additional_1.map
AnnotationLocal refinement map from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Local refinement halfmap A

Fileemd_19838_half_map_1.map
AnnotationLocal refinement halfmap A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Local refinement halfmap B

Fileemd_19838_half_map_2.map
AnnotationLocal refinement halfmap B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HSV-1 DNA polymerase-processivity factor complex in exonuclease s...

EntireName: HSV-1 DNA polymerase-processivity factor complex in exonuclease state with 1-bp DNA mismatch
Components
  • Complex: HSV-1 DNA polymerase-processivity factor complex in exonuclease state with 1-bp DNA mismatch
    • Complex: DNA polymerase-processivity factor
      • Protein or peptide: DNA polymerase catalytic subunit
    • Complex: DNA
      • DNA: DNA (46-MER)
      • DNA: DNA (67-MER)
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: HSV-1 DNA polymerase-processivity factor complex in exonuclease s...

SupramoleculeName: HSV-1 DNA polymerase-processivity factor complex in exonuclease state with 1-bp DNA mismatch
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: DNA polymerase-processivity factor

SupramoleculeName: DNA polymerase-processivity factor / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: DNA polymerase catalytic subunit

MacromoleculeName: DNA polymerase catalytic subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS
Molecular weightTheoretical: 136.683844 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MFSGGGGPLS PGGKSAARAA SGFFAPAGPR GASRGPPPCL RQNFYNPYLA PVGTQQKPTG PTQRHTYYSE CDEFRFIAPR VLDEDAPPE KRAGVHDGHL KRAPKVYCGG DERDVLRVGS GGFWPRRSRL WGGVDHAPAG FNPTVTVFHV YDILENVEHA Y GMRAAQFH ...String:
MFSGGGGPLS PGGKSAARAA SGFFAPAGPR GASRGPPPCL RQNFYNPYLA PVGTQQKPTG PTQRHTYYSE CDEFRFIAPR VLDEDAPPE KRAGVHDGHL KRAPKVYCGG DERDVLRVGS GGFWPRRSRL WGGVDHAPAG FNPTVTVFHV YDILENVEHA Y GMRAAQFH ARFMDAITPT GTVITLLGLT PEGHRVAVHV YGTRQYFYMN KEEVDRHLQC RAPRDLCERM AAALRESPGA SF RGISADH FEAEVVERTD VYYYETRPAL FYRVYVRSGR VLSYLCDNFC PAIKKYEGGV DATTRFILDN PGFVTFGWYR LKP GRNNTL AQPRAPMAFG TSSDVEFNCT ADNLAIEGGM SDLPAYKLMC FDIECKAGGE DELAFPVAGH PEDLVIQISC LLYD LSTTA LEHVLLFSLG SCDLPESHLN ELAARGLPTP VVLEFDSEFE MLLAFMTLVK QYGPEFVTGY NIINFDWPFL LAKLT DIYK VPLDGYGRMN GRGVFRVWDI GQSHFQKRSK IKVNGMVNID MYGIITDKIK LSSYKLNAVA EAVLKDKKKD LSYRDI PAY YAAGPAQRGV IGEYCIQDSL LVGQLFFKFL PHLELSAVAR LAGINITRTI YDGQQIRVFT CLLRLADQKG FILPDTQ GR FRGAGGEAPK RPAAAREDEE RPEEEGEDED EREEGGGERE PEGARETAGR HVGYQGARVL DPTSGFHVNP VVVFDFAS L YPSIIQAHNL CFSTLSLRAD AVAHLEAGKD YLEIEVGGRR LFFVKAHVRE SLLSILLRDW LAMRKQIRSR IPQSSPEEA VLLDKQQAAI KVVCNSVYGF TGVQHGLLPC LHVAATVTTI GREMLLATRE YVHARWAAFE QLLADFPEAA DMRAPGPYSM RIIYGDTDS IFVLCRGLTA AGLTAVGDKM ASHISRALFL PPIKLECEKT FTKLLLIAKK KYIGVIYGGK MLIKGVDLVR K NNCAFINR TSRALVDLLF YDDTVSGAAA ALAERPAEEW LARPLPEGLQ AFGAVLVDAH RRITDPERDI QDFVLTAELS RH PRAYTNK RLAHLTVYYK LMARRAQVPS IKDRIPYVIV AQTREVEETV ARLAALRELD AAAPGDEPAP PAALPSPAKR PRE TPSPAD PPGGASKPRK LLVSELAEDP AYAIAHGVAL NTDYYFSHLL GAACVTFKAL FGNNAKITES LLKRFIPEVW HPPD DVAAR LRTAGFGAVG AGATAEETRR MLHRAFDTLA

UniProtKB: DNA polymerase catalytic subunit

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Macromolecule #2: DNA (46-MER)

MacromoleculeName: DNA (46-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.96308 KDa
SequenceString:
(DG)(DC)(DC)(DA)(DC)(DT)(DA)(DC)(DG)(DA) (DC)(DA)(DC)(DC)(DT)(DT)(DG)(DA)(DT)(DC) (DG)(DC)(DC)(DT)(DC)(DG)(DC)(DA)(DG) (DC)(DC)(DG)(DT)(DC)(DC)(DA)(DA)(DC)(DC) (DA) (DA)(DC)(DT)(DC)(AS)(AS)

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Macromolecule #3: DNA (67-MER)

MacromoleculeName: DNA (67-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20.902295 KDa
SequenceString: (DA)(DT)(DT)(DT)(DG)(DC)(DT)(DG)(DA)(DC) (DC)(DT)(DT)(DT)(DG)(DT)(DT)(DC)(DT)(DG) (DG)(DG)(DT)(DG)(DA)(DG)(DT)(DT)(DG) (DG)(DT)(DT)(DG)(DG)(DA)(DC)(DG)(DG)(DC) (DT) (DG)(DC)(DG)(DA)(DG)(DG) ...String:
(DA)(DT)(DT)(DT)(DG)(DC)(DT)(DG)(DA)(DC) (DC)(DT)(DT)(DT)(DG)(DT)(DT)(DC)(DT)(DG) (DG)(DG)(DT)(DG)(DA)(DG)(DT)(DT)(DG) (DG)(DT)(DT)(DG)(DG)(DA)(DC)(DG)(DG)(DC) (DT) (DG)(DC)(DG)(DA)(DG)(DG)(DC)(DG) (DA)(DT)(DC)(DA)(DA)(DG)(DG)(DT)(DG)(DT) (DC)(DG) (DT)(DA)(DG)(DT)(DG)(DG)(DC)

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 7 / 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

Concentration0.4 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
5.0 mMCaCl2calcium chloride
2.0 mMC4H10O2S2DTT

Details: 20mM HEPES pH7.8, 150mM NaCl, 5mM CaCl2, 2mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 165000
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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 163943
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7luf


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-9enq:
HSV-1 DNA polymerase-processivity factor complex in exonuclease state active site with 1-bp DNA mismatch

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