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- EMDB-42887: Herpes simplex virus 1 polymerase holoenzyme bound to DNA in both... -

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Basic information

Entry
Database: EMDB / ID: EMD-42887
TitleHerpes simplex virus 1 polymerase holoenzyme bound to DNA in both open/closed conformations
Map datasharpened map after polish and CTF refinement
Sample
  • Complex: Herpes simplex virus 1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA strands
    • Protein or peptide: DNA polymerase
    • Protein or peptide: DNA polymerase processivity factor
    • DNA: Primer DNA
    • DNA: template DNA
  • Ligand: MAGNESIUM ION
  • Ligand: water
Keywordsherpes simplex virus / DNA polymerase holoenzyme / open/closed conformations / conformational dynamics / 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 processivity factor / DNA polymerase
Similarity search - Component
Biological speciesHuman herpesvirus 1 (strain KOS) / Human alphaherpesvirus 1 strain KOS / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 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_42887.png

Downloads & links

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Map

FileDownload / File: emd_42887.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map after polish and CTF refinement
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: 0.025
Minimum - Maximum-0.12296121 - 0.2296219
Average (Standard dev.)0.00003530111 (±0.0060424465)
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 1 polymerase holoenzyme UL30:UL42 in complex...

EntireName: Herpes simplex virus 1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA strands
Components
  • Complex: Herpes simplex virus 1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA strands
    • Protein or peptide: DNA polymerase
    • Protein or peptide: DNA polymerase processivity factor
    • DNA: Primer DNA
    • DNA: template DNA
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Herpes simplex virus 1 polymerase holoenzyme UL30:UL42 in complex...

SupramoleculeName: Herpes simplex virus 1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA strands
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), and one primer (3'-deoxy) DNA molecule (synthetic).
Source (natural)Organism: Human herpesvirus 1 (strain KOS)

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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 / 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 type 1 (KOS strain) processivity factor UL42 residues 1-340 preceding a maltose binding protein (MBP) tag and a PreScission protease cleavage site (MBP-PP-UL42dC340))
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS / Strain: KOS
Molecular weightTheoretical: 37.275102 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPISEFGSSR TDSPGGVAPA SHVEDASDAS LGQPEEGAPC QVVLQGAELN GILQAFAPLR TSLLDSLLVM GDRGILIHNT IFGEQVFLP LEHSQFSRYR WRGPTAAFLS LVDQKRSLLS VFRANQYPDL RRVELAITGQ APFRTLVQRI WTTTSDGEAV E LASETLMK ...String:
GPISEFGSSR TDSPGGVAPA SHVEDASDAS LGQPEEGAPC QVVLQGAELN GILQAFAPLR TSLLDSLLVM GDRGILIHNT IFGEQVFLP LEHSQFSRYR WRGPTAAFLS LVDQKRSLLS VFRANQYPDL RRVELAITGQ APFRTLVQRI WTTTSDGEAV E LASETLMK RELTSFVVLV PQGTPDVQLR LTRPQLTKVL NATGADSATP TTFELGVNGK FSVFTTSTCV TFAAREEGVS SS TSTQVQI LSNALTKAGQ AAANAKTVYG ENTHRTFSVV VDDCSMRAVL RRLQVAGGTL KFFLTTPVPS LCVTATGPNA VSA VFLLKP QKICLDWLGH SQGSPSAGSS ASR

UniProtKB: DNA polymerase processivity factor

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Macromolecule #3: Primer DNA

MacromoleculeName: Primer DNA / type: dna / ID: 3 / Details: 3'-deoxy primer DNA 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

MacromoleculeName: template DNA / type: dna / ID: 4 / Details: template DNA / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.223799 KDa
SequenceString:
(DC)(DA)(DC)(DA)(DC)(DA)(DC)(DA)(DC)(DA) (DC)(DA)(DC)(DA)(DC)(DA)(DC)(DA)(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: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 3 / 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

Concentration1.3 mg/mL
BufferpH: 7.5
Details: 25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM tris(2-carboxyethyl)phosphine (TCEP), 2 mM EDTA
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: Pelco easiGlow at 15 mA for 30 seconds under 0.39 mBar (i.e. 39 Pa)
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..
DetailsThis sample was monodisperse.

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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
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: 6173 / Average exposure time: 1.5 sec. / Average electron dose: 53.11 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: 2443151
Startup modelType of model: OTHER / Details: ab initio using Relion
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 343777
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 5 / Avg.num./class: 130467 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Detailsrigid body, minimization_global, local_grid_search, adp refinement
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 31.87 / Target criteria: correlation coefficient
Output model

PDB-8v1q:
Herpes simplex virus 1 polymerase holoenzyme bound to DNA in both open/closed conformations

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