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- EMDB-16907: HSV-1 DNA polymerase-processivity factor complex in halted elonga... -

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Basic information

Entry
Database: EMDB / ID: EMD-16907
TitleHSV-1 DNA polymerase-processivity factor complex in halted elongation state
Map data
Sample
  • Complex: HSV-1 DNA polymerase-processivity factor complex in halted elongation state
    • Protein or peptide: DNA polymerase catalytic subunit
    • Protein or peptide: DNA polymerase processivity factor
    • DNA: DNA (53-MER)
    • DNA: DNA (67-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
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 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 polymerase catalytic subunit / DNA polymerase processivity factor
Similarity search - Component
Biological speciesHuman alphaherpesvirus 1 strain KOS / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.46 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| 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, 2023-
Header (metadata) releaseApr 3, 2024-
Map releaseApr 3, 2024-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16907.png

Downloads & links

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Map

FileDownload / File: emd_16907.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 432 pix.
= 367.2 Å		0.85 Å/pix.
x 432 pix.
= 367.2 Å		0.85 Å/pix.
x 432 pix.
= 367.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.8383693 - 5.06791
Average (Standard dev.)0.0016284428 (±0.03937163)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 367.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16907_msk_1.map
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 halted elonga...

EntireName: HSV-1 DNA polymerase-processivity factor complex in halted elongation state
Components
  • Complex: HSV-1 DNA polymerase-processivity factor complex in halted elongation state
    • Protein or peptide: DNA polymerase catalytic subunit
    • Protein or peptide: DNA polymerase processivity factor
    • DNA: DNA (53-MER)
    • DNA: DNA (67-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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Supramolecule #1: HSV-1 DNA polymerase-processivity factor complex in halted elonga...

SupramoleculeName: HSV-1 DNA polymerase-processivity factor complex in halted elongation state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS

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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 polymerase processivity factor

MacromoleculeName: DNA polymerase processivity factor / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS
Molecular weightTheoretical: 51.207969 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTDSPGGVAP ASPVEDASDA SLGQPEEGAP CQVVLQGAEL NGILQAFAPL RTSLLDSLLV MGDRGILIHN TIFGEQVFLP LEHSQFSRY RWRGPTAAFL SLVDQKRSLL SVFRANQYPD LRRVELAITG QAPFRTLVQR IWTTTSDGEA VELASETLMK R ELTSFVVL ...String:
MTDSPGGVAP ASPVEDASDA SLGQPEEGAP CQVVLQGAEL NGILQAFAPL RTSLLDSLLV MGDRGILIHN TIFGEQVFLP LEHSQFSRY RWRGPTAAFL SLVDQKRSLL SVFRANQYPD LRRVELAITG QAPFRTLVQR IWTTTSDGEA VELASETLMK R ELTSFVVL VPQGTPDVQL RLTRPQLTKV LNATGADSAT PTTFELGVNG KFSVFTTSTC VTFAAREEGV SSSTSTQVQI LS NALTKAG QAAANAKTVY GENTHRTFSV VVDDCSMRAV LRRLQVGGGT LKFFLTTPVP SLCVTATGPN AVSAVFLLKP QKI CLDWLG HSQGSPSAGS SASRASGSEP TDSQDSASDA VSHGDPEDLD GAARAGEAGA LHACPMPSST TRVTPTTKRG RSGG EDARA DTALKKPKTG SPTAPPPADP VPLDTEDDSD AADGTAARPA APDARSGSRY ACYFRDLPTG EASPGAFSAF RGGPQ TPYG FGFP

UniProtKB: DNA polymerase processivity factor

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Macromolecule #3: DNA (53-MER)

MacromoleculeName: DNA (53-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 16.081343 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)(DA)(DA) ...String:
(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)(DA)(DA)(DT)(DT) (DA)(DG)(DA)(DA)(DOC)

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Macromolecule #4: DNA (67-MER)

MacromoleculeName: DNA (67-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20.845285 KDa
SequenceString: (DA)(DT)(DT)(DT)(DG)(DC)(DT)(DG)(DA)(DC) (DC)(DT)(DT)(DT)(DG)(DT)(DT)(DC)(DT)(DA) (DA)(DT)(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)(DA) (DA)(DT)(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 #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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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 mMMgCl2magnesium chloride
2.0 mMC4H10O2S2DTT

Details: 20mM HEPES pH7.8, 150mM NaCl, 5mM MgCl2, 2mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
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: 50.0 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: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
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: 2.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Details: cryoSPARC Non-uniform refinement / Number images used: 213621
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)

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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-8oj7:
HSV-1 DNA polymerase-processivity factor complex in halted elongation state

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