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- EMDB-42890: Herpes simplex virus 1 polymerase holoenzyme bound to DNA and acy... -

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Basic information

Entry
Database: EMDB / ID: EMD-42890
TitleHerpes simplex virus 1 polymerase holoenzyme bound to DNA and acyclovir triphosphate in closed conformation
Map datasharpened map
Sample
  • Complex: HERPES SIMPLEX VIRUS TYPE 1 POLYMERASE HOLOENZYME UL30:UL42 IN COMPLEX WITH dsDNA AND ACYCLOVIR TRIPHOSPHATE
    • Protein or peptide: DNA polymerase
    • Protein or peptide: DNA polymerase processivity factor
    • DNA: PRIMER DNA (32-MER)
    • DNA: TEMPLATE DNA (50-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ACYCLOVIR TRIPHOSPHATE
  • Ligand: water
Keywordsherpes simplex virus / replication / DNA polymerase / ACYCLOVIR / antiherpesvirus drug / TRANSFERASE-DNA complex
Function / homology
Function and homology information


viral DNA genome replication / 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 / DNA polymerase processivity factor
Similarity search - Component
Biological speciesHuman alphaherpesvirus 1 strain KOS / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsPan J / Abraham J / Coen DM / Shankar S / Yang P / Hogle J
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21 AI141940 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI019838 United States
CitationJournal: Cell / Year: 2024
Title: Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance.
Authors: Sundaresh Shankar / Junhua Pan / Pan Yang / Yuemin Bian / Gábor Oroszlán / Zishuo Yu / Purba Mukherjee / David J Filman / James M Hogle / Mrinal Shekhar / Donald M Coen / Jonathan Abraham /
Abstract: DNA polymerases are important drug targets, and many structural studies have captured them in distinct conformations. However, a detailed understanding of the impact of polymerase conformational ...DNA polymerases are important drug targets, and many structural studies have captured them in distinct conformations. However, a detailed understanding of the impact of polymerase conformational dynamics on drug resistance is lacking. We determined cryoelectron microscopy (cryo-EM) structures of DNA-bound herpes simplex virus polymerase holoenzyme in multiple conformations and interacting with antivirals in clinical use. These structures reveal how the catalytic subunit Pol and the processivity factor UL42 bind DNA to promote processive DNA synthesis. Unexpectedly, in the absence of an incoming nucleotide, we observed Pol in multiple conformations with the closed state sampled by the fingers domain. Drug-bound structures reveal how antivirals may selectively bind enzymes that more readily adopt the closed conformation. Molecular dynamics simulations and the cryo-EM structure of a drug-resistant mutant indicate that some resistance mutations modulate conformational dynamics rather than directly impacting drug binding, thus clarifying mechanisms that drive drug selectivity.
History
DepositionNov 21, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42890.png

Downloads & links

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Map

FileDownload / File: emd_42890.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 264. Å		0.83 Å/pix.
x 320 pix.
= 264. Å		0.83 Å/pix.
x 320 pix.
= 264. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.098319665 - 0.1670793
Average (Standard dev.)0.00001235528 (±0.0036981765)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : HERPES SIMPLEX VIRUS TYPE 1 POLYMERASE HOLOENZYME UL30:UL42 IN CO...

EntireName: HERPES SIMPLEX VIRUS TYPE 1 POLYMERASE HOLOENZYME UL30:UL42 IN COMPLEX WITH dsDNA AND ACYCLOVIR TRIPHOSPHATE
Components
  • Complex: HERPES SIMPLEX VIRUS TYPE 1 POLYMERASE HOLOENZYME UL30:UL42 IN COMPLEX WITH dsDNA AND ACYCLOVIR TRIPHOSPHATE
    • Protein or peptide: DNA polymerase
    • Protein or peptide: DNA polymerase processivity factor
    • DNA: PRIMER DNA (32-MER)
    • DNA: TEMPLATE DNA (50-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ACYCLOVIR TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: HERPES SIMPLEX VIRUS TYPE 1 POLYMERASE HOLOENZYME UL30:UL42 IN CO...

SupramoleculeName: HERPES SIMPLEX VIRUS TYPE 1 POLYMERASE HOLOENZYME UL30:UL42 IN COMPLEX WITH dsDNA AND ACYCLOVIR TRIPHOSPHATE
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: EACH COMPLEX CONSISTS OF ONE HSV-1 UL30 (expressed in insect cells), ONE HSV-1 UL42 (expressed in E.coli), ONE TEMPLATE DNA (synthetic), ONE PRIMER DNA (synthetic), AND ONE ACYCLOVIR ...Details: EACH COMPLEX CONSISTS OF ONE HSV-1 UL30 (expressed in insect cells), ONE HSV-1 UL42 (expressed in E.coli), ONE TEMPLATE DNA (synthetic), ONE PRIMER DNA (synthetic), AND ONE ACYCLOVIR TRIPHOSPHATE MOLECULE (synthetic).
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS / Strain: KOS
Molecular weightTheoretical: 197 KDa

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Macromolecule #1: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 1
Details: Herpes simplex virus type 1 (KOS strain) DNA polymerase catalytic subunit UL30 with its N-terminal 42 residues deleted and replaced by an N-terminal poly-histidine tag in the expression construct
Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS
Molecular weightTheoretical: 133.614344 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: HHHHHHNFYN PYLAPVGTQQ KPTGPTQRHT YYSECDEFRF IAPRVLDEDA PPEKRAGVHD GHLKRAPKVY CGGDERDVLR VGSGGFWPR RSRLWGGVDH APAGFNPTVT VFHVYDILEN VEHAYGMRAA QFHARFMDAI TPTGTVITLL GLTPEGHRVA V HVYGTRQY ...String:
HHHHHHNFYN PYLAPVGTQQ KPTGPTQRHT YYSECDEFRF IAPRVLDEDA PPEKRAGVHD GHLKRAPKVY CGGDERDVLR VGSGGFWPR RSRLWGGVDH APAGFNPTVT VFHVYDILEN VEHAYGMRAA QFHARFMDAI TPTGTVITLL GLTPEGHRVA V HVYGTRQY FYMNKEEVDR HLQCRAPRDL CERMAAALRE SPGASFRGIS ADHFEAEVVE RTDVYYYETR PALFYRVYVR SG RVLSYLC DNFCPAIKKY EGGVDATTRF ILDNPGFVTF GWYRLKPGRN NTLAQPRAPM AFGTSSDVEF NCTADNLAIE GGM SDLPAY KLMCFDIECK AGGEDELAFP VAGHPEDLVI QISCLLYDLS TTALEHVLLF SLGSCDLPES HLNELAARGL PTPV VLEFD SEFEMLLAFM TLVKQYGPEF VTGYNIINFD WPFLLAKLTD IYKVPLDGYG RMNGRGVFRV WDIGQSHFQK RSKIK VNGM VNIDMYGIIT DKIKLSSYKL NAVAEAVLKD KKKDLSYRDI PAYYATGPAQ RGVIGEYCIQ DSLLVGQLFF KFLPHL ELS AVARLAGINI TRTIYDGQQI RVFTCLLRLA DQKGFILPDT QGRFRGAGGE APKRPAAARE DEERPEEEGE DEDEREE GG GEREPEGARE TAGRHVGYQG ARVLDPTSGF HVNPVVVFDF ASLYPSIIQA HNLCFSTLSL RADAVAHLEA GKDYLEIE V GGRRLFFVKA HVRESLLSIL LRDWLAMRKQ IRSRIPQSSP EEAVLLDKQQ AAIKVVCNSV YGFTGVQHGL LPCLHVAAT VTTIGREMLL ATREYVHARW AAFEQLLADF PEAADMRAPG PYSMRIIYGD TDSIFVLCRG LTAAGLTAMG DKMASHISRA LFLPPIKLE CEKTFTKLLL IAKKKYIGVI YGGKMLIKGV DLVRKNNCAF INRTSRALVD LLFYDDTVSG AAAALAERPA E EWLARPLP EGLQAFGAVL VDAHRRITDP ERDIQDFVLT AELSRHPRAY TNKRLAHLTV YYKLMARRAQ VPSIKDRIPY VI VAQTREV EETVARLAAL RELDAAAPGD EPAPPAALPS PAKRPRETPS HADPPGGASK PRKLLVSELA EDPAYAIAHG VAL NTDYYF SHLLGAACVT FKALFGNNAK ITESLLKRFI PEVWHPPDDV AARLRAAGFG AVGAGATAEE TRRMLHRAFD TLA

UniProtKB: DNA polymerase

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Macromolecule #2: DNA polymerase processivity factor

MacromoleculeName: DNA polymerase processivity factor / type: protein_or_peptide / ID: 2
Details: Herpes simplex virus 1 (KOS strain) DNA polymerase processivity factor UL42 residues 1-340 tagged with a prescission protease cleavage site followed by a maltose binding proten (MBP) tag in ...Details: Herpes simplex virus 1 (KOS strain) DNA polymerase processivity factor UL42 residues 1-340 tagged with a prescission protease cleavage site followed by a maltose binding proten (MBP) tag in the expression construct
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS
Molecular weightTheoretical: 36.34618 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 LRRLQVAGGT LKFFLTTPVP SLCVTATGPN AVSAVFLLKP QKI CLDWLG HSQGSPSAGS SASR

UniProtKB: DNA polymerase processivity factor

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Macromolecule #3: PRIMER DNA (32-MER)

MacromoleculeName: PRIMER DNA (32-MER) / type: dna / ID: 3
Details: 3'-deoxyl primer DNA strand, no mismatch with template strand
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.866353 KDa
SequenceString:
(DG)(DA)(DT)(DT)(DA)(DC)(DG)(DA)(DA)(DT) (DT)(DC)(DG)(DA)(DG)(DC)(DT)(DC)(DG)(DG) (DT)(DA)(DC)(DC)(DC)(DG)(DG)(DG)(DG) (DA)(DT)(DOC)

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Macromolecule #4: TEMPLATE DNA (50-MER)

MacromoleculeName: TEMPLATE DNA (50-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.199773 KDa
SequenceString:
(DC)(DA)(DC)(DA)(DC)(DA)(DC)(DA)(DC)(DA) (DC)(DA)(DC)(DA)(DC)(DA)(DC)(DC)(DG)(DA) (DT)(DC)(DC)(DC)(DC)(DG)(DG)(DG)(DT) (DA)(DC)(DC)(DG)(DA)(DG)(DC)(DT)(DC)(DG) (DA) (DA)(DT)(DT)(DC)(DG)(DT)(DA)(DA) (DT)(DC)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ACYCLOVIR TRIPHOSPHATE

MacromoleculeName: ACYCLOVIR TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: AVP
Molecular weightTheoretical: 465.144 Da
Chemical component information

ChemComp-AVP:
ACYCLOVIR TRIPHOSPHATE

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 4 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration1.38 mg/mL
BufferpH: 7.5
Details: 25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM tris(2-carboxyethyl)phosphine (TCEP)
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
Details: GRIDS WERE GLOW DISCHARGED IN A PELCO EASIGLOW AT 15 MA FOR 30 SECONDS UNDER 0.39 MBAR.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: 3 MICROLITERS OF SAMPLE WERE BLOTTED FOR 3 SECONDS WITH FILTER PAPER SATURATED UNDER 100% HUMIDITY PRIOR TO PLUNGING..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
SoftwareName: SerialEM (ver. 3.8)
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3268 / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 60606 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 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

Particle selectionNumber selected: 1214260
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 187300
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 5 / Avg.num./class: 59848 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: UCSF Chimera (ver. 1.13.1)
DetailsRIGID BODY, MINIMIZATION_GLOBAL, LOCAL_GRID_SEARCH, ADP REFINEMENT
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 33.64 / Target criteria: CORRELATION COEFFICIENT
Output model

PDB-8v1t:
Herpes simplex virus 1 polymerase holoenzyme bound to DNA and acyclovir triphosphate in closed conformation

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