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- PDB-8ojc: HSV-1 DNA polymerase active site in alternative exonuclease state -

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Basic information

Entry
Database: PDB / ID: 8ojc
TitleHSV-1 DNA polymerase active site in alternative exonuclease state
Components
  • DNA (47-MER)
  • DNA polymerase catalytic subunit
KeywordsTRANSFERASE / DNA / Polymerase / Complex
Function / homology
Function and homology information


DNA polymerase activity / DNA polymerase complex / 5'-3' exonuclease activity / bidirectional double-stranded viral DNA replication / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / 3'-5'-DNA exonuclease activity / ribonuclease H / SOS response / base-excision repair, gap-filling ...DNA polymerase activity / DNA polymerase complex / 5'-3' exonuclease activity / bidirectional double-stranded viral DNA replication / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / 3'-5'-DNA exonuclease activity / ribonuclease H / SOS response / base-excision repair, gap-filling / 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 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-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily ...DNA polymerase catalytic subunit Pol, C-terminal / DNA polymerase catalytic subunit Pol / DNA polymerase family B, thumb 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-directed DNA polymerase, family B, multifunctional 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 / DNA (> 10) / DNA polymerase catalytic subunit
Similarity search - Component
Biological speciesHuman alphaherpesvirus 1 strain KOS
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.08 Å
AuthorsGustavsson, E. / Grunewald, K. / Elias, P. / Hallberg, B.M.
Funding support Sweden, Germany, 2items
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 24, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: DNA (47-MER)
A: DNA polymerase catalytic subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)151,0244
Polymers150,9442
Non-polymers802
Water5,188288
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: DNA chain DNA (47-MER)


Mass: 14260.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein DNA polymerase catalytic subunit


Mass: 136683.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human alphaherpesvirus 1 strain KOS / Gene: UL30 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P04293, DNA-directed DNA polymerase, ribonuclease H
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 288 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: HSV-1 DNA polymerase-processivity factor complex in exonuclease state
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.8 / Details: 20mM HEPES pH7.8, 150mM NaCl, 5mM CaCl2, 2mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
35 mMcalcium chlorideCaCl21
42 mMDTTC4H10O2S21
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF ChimeraXmodel fitting
9Cootmodel refinement
10cryoSPARC3.3initial Euler assignment
11cryoSPARC4final Euler assignment
13cryoSPARC43D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 2.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48633 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7LUF

7luf


Accession code: 7LUF / Source name: PDB / Type: experimental model
RefinementResolution: 2.08→2.08 Å / Cor.coef. Fo:Fc: 0.757 / WRfactor Rwork: 0.335 / SU B: 8.663 / SU ML: 0.183 / Average fsc free: 0 / Average fsc overall: 0.829 / Average fsc work: 0.829 / ESU R: 0.188
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3352 77843 -
all0.335 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 43.718 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0123297
ELECTRON MICROSCOPYr_bond_other_d00.0163074
ELECTRON MICROSCOPYr_angle_refined_deg1.3231.6494485
ELECTRON MICROSCOPYr_angle_other_deg0.4421.5727075
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.7165397
ELECTRON MICROSCOPYr_dihedral_angle_2_deg4.906522
ELECTRON MICROSCOPYr_dihedral_angle_other_2_deg0.067103
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.63510529
ELECTRON MICROSCOPYr_dihedral_angle_6_deg15.97810149
ELECTRON MICROSCOPYr_chiral_restr0.0680.2490
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.023811
ELECTRON MICROSCOPYr_gen_planes_other0.0010.02768
ELECTRON MICROSCOPYr_nbd_refined0.1990.2617
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1880.22818
ELECTRON MICROSCOPYr_nbtor_refined0.1770.21548
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0770.21668
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1790.2196
ELECTRON MICROSCOPYr_metal_ion_refined0.2650.24
ELECTRON MICROSCOPYr_mcbond_it5.4094.331592
ELECTRON MICROSCOPYr_mcbond_other5.414.3251591
ELECTRON MICROSCOPYr_mcangle_it9.4697.7441987
ELECTRON MICROSCOPYr_mcangle_other9.4677.7491988
ELECTRON MICROSCOPYr_scbond_it5.965.1511705
ELECTRON MICROSCOPYr_scbond_other5.9585.1561706
ELECTRON MICROSCOPYr_scangle_it10.4799.1282498
ELECTRON MICROSCOPYr_scangle_other10.4779.1312499
ELECTRON MICROSCOPYr_lrange_it19.44549.34513970
ELECTRON MICROSCOPYr_lrange_other19.26449.213694
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: 0 / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
2.4-2.4620.47657740.47657740.6690.476
2.462-2.530.44955610.44955610.6980.449
2.53-2.6030.42954710.42954710.7180.429
2.603-2.6830.39352910.39352910.7520.393
2.683-2.7710.3651710.3651710.7860.36
2.771-2.8680.33949720.33949720.8190.339
2.868-2.9760.31848250.31848250.8490.318
2.976-3.0980.31445450.31445450.8620.314
3.098-3.2350.344300.344300.880.3
3.235-3.3930.2842170.2842170.9020.28
3.393-3.5760.26440500.26440500.9180.264
3.576-3.7930.25338220.25338220.9260.253
3.793-4.0540.25935550.25935550.930.259
4.054-4.3780.26933500.26933500.9310.269
4.378-4.7950.24330460.24330460.9320.243
4.795-5.3580.23627680.23627680.9240.236
5.358-6.1830.3724590.3724590.8610.37
6.183-7.5640.49820330.49820330.8370.498
7.564-10.6560.45216130.45216130.8140.452
10.656-86.9020.7448900.7448900.9250.744

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