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- EMDB-42891: Herpes simplex virus 1 polymerase W781V mutant holoenzyme bound t... -

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Basic information

Entry
Database: EMDB / ID: EMD-42891
TitleHerpes simplex virus 1 polymerase W781V mutant holoenzyme bound to DNA in editing conformation
Map datasharpened map after polishing
Sample
  • Complex: HSV-1 DNA polymerase W781V mutant holoenzyme in editing complex
    • Protein or peptide: DNA polymerase
    • Protein or peptide: DNA polymerase processivity factor
    • DNA: DNA primer
    • DNA: DNA template
Keywordsherpes simplex virus / replication / DNA polymerase holoenzyme / W781V / editing conformation / TRANSFERASE-DNA complex / VIRAL PROTEIN
Function / homology: / :
Function and homology information
Biological speciesHuman herpesvirus 1 (strain KOS) / Human alphaherpesvirus 1 strain KOS / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.1 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-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42891.png

Downloads & links

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Map

FileDownload / File: emd_42891.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map after polishing
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.65 Å/pix.
x 160 pix.
= 264. Å		1.65 Å/pix.
x 160 pix.
= 264. Å		1.65 Å/pix.
x 160 pix.
= 264. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.10176018 - 0.22815637
Average (Standard dev.)0.00034529108 (±0.007816389)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
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 : HSV-1 DNA polymerase W781V mutant holoenzyme in editing complex

EntireName: HSV-1 DNA polymerase W781V mutant holoenzyme in editing complex
Components
  • Complex: HSV-1 DNA polymerase W781V mutant holoenzyme in editing complex
    • Protein or peptide: DNA polymerase
    • Protein or peptide: DNA polymerase processivity factor
    • DNA: DNA primer
    • DNA: DNA template

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Supramolecule #1: HSV-1 DNA polymerase W781V mutant holoenzyme in editing complex

SupramoleculeName: HSV-1 DNA polymerase W781V mutant holoenzyme in editing complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Herpes simplex virus type 1 KOS strain polymerase W781V mutant holoenzyme UL30W781V:UL42 in complex with primer DNA that has a 3'-end mismatch with template DNA
Source (natural)Organism: Human herpesvirus 1 (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 UL30 with its N-terminal 42 residues deleted and replaced by an N-terminal poly-histidine tag in the expression construct
Enantiomer: LEVO
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS / Strain: KOS
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: HHHHHHNFYN PYLAPVGTQQ KPTGPTQRHT YYSECDEFRF IAPRVLDEDA PPEKRAGVHD GHLKRAPKVY CGGDERDVLR VGSGGFWPRR SRLWGGVDHA PAGFNPTVTV FHVYDILENV EHAYGMRAAQ FHARFMDAIT PTGTVITLLG LTPEGHRVAV HVYGTRQYFY ...String:
HHHHHHNFYN PYLAPVGTQQ KPTGPTQRHT YYSECDEFRF IAPRVLDEDA PPEKRAGVHD GHLKRAPKVY CGGDERDVLR VGSGGFWPRR SRLWGGVDHA PAGFNPTVTV FHVYDILENV EHAYGMRAAQ FHARFMDAIT PTGTVITLLG LTPEGHRVAV HVYGTRQYFY MNKEEVDRHL QCRAPRDLCE RMAAALRESP GASFRGISAD HFEAEVVERT DVYYYETRPA LFYRVYVRSG RVLSYLCDNF CPAIKKYEGG VDATTRFILD NPGFVTFGWY RLKPGRNNTL AQPRAPMAFG TSSDVEFNCT ADNLAIEGGM SDLPAYKLMC FDIECKAGGE DELAFPVAGH PEDLVIQISC LLYDLSTTAL EHVLLFSLGS CDLPESHLNE LAARGLPTPV VLEFDSEFEM LLAFMTLVKQ YGPEFVTGYN IINFDWPFLL AKLTDIYKVP LDGYGRMNGR GVFRVWDIGQ SHFQKRSKIK VNGMVNIDMY GIITDKIKLS SYKLNAVAEA VLKDKKKDLS YRDIPAYYAT GPAQRGVIGE YCIQDSLLVG QLFFKFLPHL ELSAVARLAG INITRTIYDG QQIRVFTCLL RLADQKGFIL PDTQGRFRGA GGEAPKRPAA AREDEERPEE EGEDEDEREE GGGEREPEGA RETAGRHVGY QGARVLDPTS GFHVNPVVVF DFASLYPSII QAHNLCFSTL SLRADAVAHL EAGKDYLEIE VGGRRLFFVK AHVRESLLSI LLRDVLAMRK QIRSRIPQSS PEEAVLLDKQ QAAIKVVCNS VYGFTGVQHG LLPCLHVAAT VTTIGREMLL ATREYVHARW AAFEQLLADF PEAADMRAPG PYSMRIIYGD TDSIFVLCRG LTAAGLTAMG DKMASHISRA LFLPPIKLEC EKTFTKLLLI AKKKYIGVIY GGKMLIKGVD LVRKNNCAFI NRTSRALVDL LFYDDTVSGA AAALAERPAE EWLARPLPEG LQAFGAVLVD AHRRITDPER DIQDFVLTAE LSRHPRAYTN KRLAHLTVYY KLMARRAQVP SIKDRIPYVI VAQTREVEET VARLAALREL DAAAPGDEPA PPAALPSPAK RPRETPSHAD PPGGASKPRK LLVSELAEDP AYAIAHGVAL NTDYYFSHLL GAACVTFKAL FGNNAKITES LLKRFIPEVW HPPDDVAARL RAAGFGAVGA GATAEETRRM LHRAFDTLA

UniProtKB: UNIPROTKB: AFE62858

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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))
Enantiomer: LEVO
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTDSPGGVAP ASPVEDASDA SLGQPEEGAP CQVVLQGAEL NGILQAFAPL RTSLLDSLLV MGDRGILIHN TIFGEQVFLP LEHSQFSRYR WRGPTAAFLS LVDQKRSLLS VFRANQYPDL RRVELAITGQ APFRTLVQRI WTTTSDGEAV ELASETLMKR ELTSFVVLVP ...String:
MTDSPGGVAP ASPVEDASDA SLGQPEEGAP CQVVLQGAEL NGILQAFAPL RTSLLDSLLV MGDRGILIHN TIFGEQVFLP LEHSQFSRYR WRGPTAAFLS LVDQKRSLLS VFRANQYPDL RRVELAITGQ APFRTLVQRI WTTTSDGEAV ELASETLMKR ELTSFVVLVP QGTPDVQLRL TRPQLTKVLN ATGADSATPT TFELGVNGKF SVFTTSTCVT FAAREEGVSS STSTQVQILS NALTKAGQAA ANAKTVYGEN THRTFSVVVD DCSMRAVLRR LQVAGGTLKF FLTTPVPSLC VTATGPNAVS AVFLLKPQKI CLDWLGHSQG SPSAGSSASR

UniProtKB: UNIPROTKB: AFE62870

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Macromolecule #3: DNA primer

MacromoleculeName: DNA primer / type: dna / ID: 3 / Details: 3'-deoxy primer DNA strand / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
GATTACGAAT TCGAGCTCGG TACCCGGGGA T(DOC)

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Macromolecule #4: DNA template

MacromoleculeName: DNA template / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
CACACACACA CACACACAGA TCCCCGGGTA CCGAGCTCGA ATTCGTAATC

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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: 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: 1678 / Average exposure time: 1.5 sec. / Average electron dose: 58.4 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: 972931
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: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 232946
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: 194586 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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