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- PDB-9enq: HSV-1 DNA polymerase-processivity factor complex in exonuclease s... -

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Basic information

Entry
Database: PDB / ID: 9enq
TitleHSV-1 DNA polymerase-processivity factor complex in exonuclease state active site with 1-bp DNA mismatch
Components
  • DNA (46-MER)
  • DNA (67-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: 2.12 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 13, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 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
A: DNA polymerase catalytic subunit
C: DNA (46-MER)
D: DNA (67-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,6295
Polymers171,5493
Non-polymers802
Water1267
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#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: DNA chain DNA (46-MER)


Mass: 13963.080 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (67-MER)


Mass: 20902.295 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-CA / CALCIUM ION


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


Mass: 18.015 Da / Num. of mol.: 7 / 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

Component
IDNameTypeEntity IDParent-IDSource
1HSV-1 DNA polymerase-processivity factor complex in exonuclease state with 1-bp DNA mismatchCOMPLEX#1-#30RECOMBINANT
2DNA polymerase-processivity factorCOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Human alphaherpesvirus 1 strain KOS10306
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33synthetic construct (others)32630
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: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
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
1cryoSPARC4.4particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7Coot0.9.8.92model fitting
9Coot0.9.8.92model refinement
10cryoSPARC4.4initial Euler assignment
11cryoSPARC4.4final Euler assignment
13cryoSPARC4.43D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 2.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163943 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7LUF

7luf


Accession code: 7LUF / Source name: PDB / Type: experimental model
RefinementResolution: 2.12→2.12 Å / Cor.coef. Fo:Fc: 0.885 / WRfactor Rwork: 0.316 / SU B: 3.974 / SU ML: 0.092 / Average fsc free: 0 / Average fsc overall: 0.8025 / Average fsc work: 0.8025 / ESU R: 0.117
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3158 206333 -
all0.316 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 57.716 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0125884
ELECTRON MICROSCOPYr_bond_other_d00.0165495
ELECTRON MICROSCOPYr_angle_refined_deg1.4191.8338003
ELECTRON MICROSCOPYr_angle_other_deg0.51.74912619
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.225708
ELECTRON MICROSCOPYr_dihedral_angle_2_deg8.495549
ELECTRON MICROSCOPYr_dihedral_angle_other_2_deg10.18913.5717
ELECTRON MICROSCOPYr_dihedral_angle_3_deg10.94810941
ELECTRON MICROSCOPYr_dihedral_angle_6_deg15.18910268
ELECTRON MICROSCOPYr_chiral_restr0.0750.2878
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.026925
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021415
ELECTRON MICROSCOPYr_nbd_refined0.2010.2845
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1740.24449
ELECTRON MICROSCOPYr_nbtor_refined0.1680.22769
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0710.22895
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1320.279
ELECTRON MICROSCOPYr_metal_ion_refined0.1020.22
ELECTRON MICROSCOPYr_mcbond_it4.1955.1282836
ELECTRON MICROSCOPYr_mcbond_other4.1955.1282836
ELECTRON MICROSCOPYr_mcangle_it6.9049.2653543
ELECTRON MICROSCOPYr_mcangle_other6.9039.2643544
ELECTRON MICROSCOPYr_scbond_it5.5216.5263048
ELECTRON MICROSCOPYr_scbond_other5.5176.5263048
ELECTRON MICROSCOPYr_scangle_it9.46411.6284460
ELECTRON MICROSCOPYr_scangle_other9.46311.6334461
ELECTRON MICROSCOPYr_lrange_it16.31365.05723742
ELECTRON MICROSCOPYr_lrange_other16.30165.06423734
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
2.1-2.1550.777152840.777152840.470.777
2.155-2.2140.543148810.543148810.5680.543
2.214-2.2780.498144570.498144570.6320.498
2.278-2.3480.459140210.459140210.7050.459
2.348-2.4250.429137040.429137040.7410.429
2.425-2.510.374131200.374131200.7930.374
2.51-2.6050.333127220.333127220.8380.333
2.605-2.7110.309121490.309121490.8620.309
2.711-2.8310.298117780.298117780.890.298
2.831-2.9690.292112570.292112570.9150.292
2.969-3.130.29106470.29106470.9220.29
3.13-3.320.265100930.265100930.9370.265
3.32-3.5490.24894350.24894350.9460.248
3.549-3.8330.23588540.23588540.9480.235
3.833-4.1980.21381090.21381090.9530.213
4.198-4.6930.20173090.20173090.9550.201
4.693-5.4170.264980.264980.9490.2
5.417-6.6320.33254470.33254470.9060.332
6.632-9.3640.36142400.36142400.8960.361
9.364-1200.47423280.47423280.9620.474

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