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- PDB-8ojb: HSV-1 DNA polymerase-processivity factor complex in exonuclease s... -

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Basic information

Entry
Database: PDB / ID: 8ojb
TitleHSV-1 DNA polymerase-processivity factor complex in exonuclease state active site
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 / ribonuclease H / RNA-DNA hybrid ribonuclease activity / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding ...DNA polymerase activity / DNA polymerase complex / 5'-3' exonuclease activity / bidirectional double-stranded viral DNA replication / ribonuclease H / 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 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: 1.9 Å
AuthorsGustavsson, E. / Grunewald, K. / Elias, P. / Hallberg, B.M.
Funding support Sweden, Germany, 6items
OrganizationGrant numberCountry
Swedish Research Council2017-06702 Sweden
German Research Foundation (DFG)152/772-1 Germany
German Research Foundation (DFG)152/774-1 Germany
German Research Foundation (DFG)152/775-1 Germany
German Research Foundation (DFG)152/776-1 Germany
German Research Foundation (DFG)152/777-1 Germany
CitationJournal: To Be Published
Title: Dynamics of the Herpes simplex virus DNA polymerase holoenzyme during DNA synthesis and proof-reading revealed by Cryo-EM
Authors: Gustavsson, E. / Grunewald, K. / Elias, P. / Hallberg, B.M.
History
DepositionMar 24, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release

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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
Water2,090116
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.: 116 / 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: #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: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142948 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7LUF

7luf


Accession code: 7LUF / Source name: PDB / Type: experimental model
RefinementResolution: 1.9→1.9 Å / Cor.coef. Fo:Fc: 0.611 / WRfactor Rwork: 0.385 / SU B: 1.946 / SU ML: 0.048 / Average fsc free: 0 / Average fsc overall: 0.7807 / Average fsc work: 0.7807 / ESU R: 0.053
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3852 1277242 -
all0.385 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 35.646 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0126838
ELECTRON MICROSCOPYr_bond_other_d00.0166469
ELECTRON MICROSCOPYr_angle_refined_deg1.4061.6519285
ELECTRON MICROSCOPYr_angle_other_deg0.4751.56914859
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.5565837
ELECTRON MICROSCOPYr_dihedral_angle_2_deg6.56556
ELECTRON MICROSCOPYr_dihedral_angle_other_2_deg1.72103
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.002101119
ELECTRON MICROSCOPYr_dihedral_angle_6_deg16.3210307
ELECTRON MICROSCOPYr_chiral_restr0.070.21021
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.028017
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021638
ELECTRON MICROSCOPYr_nbd_refined0.210.21246
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1870.25799
ELECTRON MICROSCOPYr_nbtor_refined0.1850.23377
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0780.23775
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1460.2192
ELECTRON MICROSCOPYr_metal_ion_refined0.2590.23
ELECTRON MICROSCOPYr_mcbond_it4.4243.2233344
ELECTRON MICROSCOPYr_mcbond_other4.4243.2233344
ELECTRON MICROSCOPYr_mcangle_it7.9845.7964181
ELECTRON MICROSCOPYr_mcangle_other7.9835.7984182
ELECTRON MICROSCOPYr_scbond_it4.3623.9783494
ELECTRON MICROSCOPYr_scbond_other4.3623.9813495
ELECTRON MICROSCOPYr_scangle_it7.8586.9475104
ELECTRON MICROSCOPYr_scangle_other7.8576.9495105
ELECTRON MICROSCOPYr_lrange_it16.24937.08328635
ELECTRON MICROSCOPYr_lrange_other16.25637.10128583
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
1.87-1.9193.091948333.091948330.1713.091
1.919-1.9711.169920631.169920630.451.169
1.971-2.0280.453893740.453893740.6670.453
2.028-2.0910.388871220.388871220.7510.388
2.091-2.1590.369843130.369843130.7940.369
2.159-2.2350.369815250.369815250.8280.369
2.235-2.3190.362787630.362787630.8520.362
2.319-2.4140.345755360.345755360.870.345
2.414-2.5210.332729020.332729020.8840.332
2.521-2.6450.31692380.31692380.8980.31
2.645-2.7880.286659490.286659490.9080.286
2.788-2.9570.257625540.257625540.9260.257
2.957-3.1610.237587150.237587150.9340.237
3.161-3.4140.207544210.207544210.9470.207
3.414-3.740.189502980.189502980.9540.189
3.74-4.1810.185453180.185453180.9560.185
4.181-4.8280.192401320.192401320.9560.192
4.828-5.9130.304337460.304337460.9340.304
5.913-8.3610.545261540.545261540.8820.545
8.361-353.50.82142900.82142900.850.82

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