[English] 日本語
Yorodumi
- EMDB-28664: Herpes simplex virus 1 DNA polymerase holoenzyme bound to DNA tem... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-28664
TitleHerpes simplex virus 1 DNA polymerase holoenzyme bound to DNA template and primer, dNTP-free (editing mode)
Map dataHSV-1 polymerase holoenzyme bound by template and primer DNA, dNTP-free sample (editing conformation)
Sample
  • Complex: HSV-1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA (no mismatch), dNTP free
    • Protein or peptide: HSV-1 UL30
    • Protein or peptide: HSV-1 UL42
    • DNA: Template DNA (50-mer)
    • DNA: Primer DNA (32-mer)
Keywordsherpes simplex virus / replication / DNA polymerase / holoenzyme / editing complex / VIRAL PROTEIN-DNA complex
Biological speciesHuman herpesvirus 1 (strain KOS) / Human alphaherpesvirus 1 strain KOS / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.77 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
DepositionOct 26, 2022-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_28664.png

Downloads & links

-
Map

FileDownload / File: emd_28664.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHSV-1 polymerase holoenzyme bound by template and primer DNA, dNTP-free sample (editing conformation)
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: 0.008
Minimum - Maximum-0.02600899 - 0.053269174
Average (Standard dev.)-0.00004565547 (±0.0017838922)
SymmetrySpace group: 1
Details

EMDB XML:

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

-
Supplemental data

-
Sample components

-
Entire : HSV-1 polymerase holoenzyme UL30:UL42 in complex with template an...

EntireName: HSV-1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA (no mismatch), dNTP free
Components
  • Complex: HSV-1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA (no mismatch), dNTP free
    • Protein or peptide: HSV-1 UL30
    • Protein or peptide: HSV-1 UL42
    • DNA: Template DNA (50-mer)
    • DNA: Primer DNA (32-mer)

-
Supramolecule #1: HSV-1 polymerase holoenzyme UL30:UL42 in complex with template an...

SupramoleculeName: HSV-1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA (no mismatch), dNTP free
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Herpes simplex virus type 1 polymerase holoenzyme UL30:UL42 in complex with template and primer DNA strands (no mismatch), dNTP free
Source (natural)Organism: Human herpesvirus 1 (strain KOS)
Molecular weightTheoretical: 197 KDa

-
Macromolecule #1: HSV-1 UL30

MacromoleculeName: HSV-1 UL30 / 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
Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
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 LLRDWLAMRK 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

GENBANK: GENBANK: AFE62858

-
Macromolecule #2: HSV-1 UL42

MacromoleculeName: HSV-1 UL42 / type: protein_or_peptide / ID: 2
Details: Herpes simplex virus type 1 DNA polymerase processivity factor UL42 residues 1-340 (UL42delC340) followed by a PreScission protease cleavage site and a maltose binding protein (MBP) tag
Enantiomer: LEVO
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS / Strain: KOS
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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

GENBANK: GENBANK: AFE62870

-
Macromolecule #3: Template DNA (50-mer)

MacromoleculeName: Template DNA (50-mer) / type: dna / ID: 3 / Details: tempalte DNA / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
CACACACACA CACACACAGA TCCCCGGGTA CCGAGCTCGA ATTCGTAATC

-
Macromolecule #4: Primer DNA (32-mer)

MacromoleculeName: Primer DNA (32-mer) / type: dna / ID: 4 / Details: 3'-deoxyl primer DNA strand / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
GATTACGAAT TCGAGCTCGG TACCCGGGGA T(DOC)

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
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)
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 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.

-
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

+
Image processing

Particle selectionNumber selected: 2443151
Startup modelType of model: OTHER / Details: ab initio using Relion
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 214544
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)

-
Atomic model buiding 1

Detailsrigid body, minimization_global, local_grid_search, adp refinement
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 45.88 / Target criteria: correlation coefficient

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbjlvh1.pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more