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- PDB-8oja: HSV-1 DNA polymerase-processivity factor complex in exonuclease state -

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Basic information

Entry
Database: PDB / ID: 8oja
TitleHSV-1 DNA polymerase-processivity factor complex in exonuclease state
Components
  • (DNA polymerase ...) x 2
  • DNA (47-MER)
  • DNA (68-MER)
KeywordsTRANSFERASE / DNA / Polymerase / Complex
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 polymerase processivity factor activity / DNA-templated DNA replication / RNA-DNA hybrid ribonuclease activity / DNA replication / DNA-directed DNA polymerase ...DNA polymerase activity / DNA polymerase complex / bidirectional double-stranded viral DNA replication / 5'-3' exonuclease activity / ribonuclease H / DNA polymerase processivity factor activity / DNA-templated DNA replication / RNA-DNA hybrid ribonuclease activity / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / host cell nucleus / DNA binding
Similarity search - Function
DNA polymerase processivity factor (UL42) / DNA polymerase processivity factor (UL42) / 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 processivity factor (UL42) / DNA polymerase processivity factor (UL42) / 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 / DNA (> 10) / DNA polymerase catalytic subunit / DNA polymerase processivity factor
Similarity search - Component
Biological speciesHuman alphaherpesvirus 1 strain KOS
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.87 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
Revision 1.2Jul 17, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase catalytic subunit
B: DNA polymerase processivity factor
C: DNA (47-MER)
D: DNA (68-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)223,4646
Polymers223,3844
Non-polymers802
Water3,171176
1


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

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Components

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DNA polymerase ... , 2 types, 2 molecules AB

#1: 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
#2: Protein DNA polymerase processivity factor / DNA-binding protein UL42 / Polymerase accessory protein / PAP


Mass: 51207.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human alphaherpesvirus 1 strain KOS / Gene: UL42 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P10226

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain DNA (47-MER)


Mass: 14260.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (68-MER)


Mass: 21231.502 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 178 molecules

#5: Chemical ChemComp-CA / CALCIUM ION


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


Mass: 18.015 Da / Num. of mol.: 176 / Source method: isolated from a natural source / Formula: H2O

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Details

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, #4 / 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 chlorideNaCl1
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 FIELD / 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
9cryoSPARC3.3initial Euler assignment
10cryoSPARC4final Euler assignment
12cryoSPARC43D reconstruction
13Cootmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 1.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 539028 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7LUF

7luf


Accession code: 7LUF / Source name: PDB / Type: experimental model
RefinementResolution: 1.87→353.5 Å / Cor.coef. Fo:Fc: 0.878 / WRfactor Rwork: 0.33 / SU B: 2.752 / SU ML: 0.071 / Average fsc free: 0 / Average fsc overall: 0.8503 / Average fsc work: 0.8503 / ESU R: 0.062
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3302 1224442 -
all0.33 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 62.864 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.01111973
ELECTRON MICROSCOPYr_bond_other_d00.01610925
ELECTRON MICROSCOPYr_angle_refined_deg1.3051.66716477
ELECTRON MICROSCOPYr_angle_other_deg0.4331.56625092
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.3951336
ELECTRON MICROSCOPYr_dihedral_angle_2_deg5.368599
ELECTRON MICROSCOPYr_dihedral_angle_other_2_deg0.4376.2544
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.74101772
ELECTRON MICROSCOPYr_dihedral_angle_6_deg16.2110480
ELECTRON MICROSCOPYr_chiral_restr0.0930.21864
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0213367
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022783
ELECTRON MICROSCOPYr_nbd_refined0.2210.22291
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1980.29740
ELECTRON MICROSCOPYr_nbtor_refined0.1810.25625
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0750.26006
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1770.2320
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_other0.0860.21
ELECTRON MICROSCOPYr_metal_ion_refined0.1940.23
ELECTRON MICROSCOPYr_mcbond_it4.6494.6255358
ELECTRON MICROSCOPYr_mcbond_other4.6494.6255358
ELECTRON MICROSCOPYr_mcangle_it7.5868.336690
ELECTRON MICROSCOPYr_mcangle_other7.5868.3336691
ELECTRON MICROSCOPYr_scbond_it5.2268.6766615
ELECTRON MICROSCOPYr_scbond_other5.2268.6766615
ELECTRON MICROSCOPYr_scangle_it9.15115.7929787
ELECTRON MICROSCOPYr_scangle_other9.15115.7999788
ELECTRON MICROSCOPYr_lrange_it18.66577.67949624
ELECTRON MICROSCOPYr_lrange_other18.67677.80549517
LS refinement shell

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

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
1.87-1.9191.156906801.156906800.6521.156
1.919-1.9710.736880300.736880300.7040.736
1.971-2.0280.379860300.379860300.7950.379
2.028-2.0910.326837380.326837380.8360.326
2.091-2.1590.316806690.316806690.8510.316
2.159-2.2350.324781920.324781920.8630.324
2.235-2.3190.318754420.318754420.8740.318
2.319-2.4140.308725140.308725140.8820.308
2.414-2.5210.327696670.327696670.8790.327
2.521-2.6450.33664790.33664790.8780.33
2.645-2.7880.316633090.316633090.8870.316
2.788-2.9570.295598580.295598580.90.295
2.957-3.1610.273562560.273562560.910.273
3.161-3.4140.25522610.25522610.9230.25
3.414-3.740.227482780.227482780.9350.227
3.74-4.1810.217434180.217434180.9390.217
4.181-4.8280.213383760.213383760.9430.213
4.828-5.9130.301325080.301325080.9210.301
5.913-8.3610.439250110.439250110.8750.439
8.361-353.50.315137280.315137280.9170.315

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