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- EMDB-16925: Focused refinement of HSV-1 DNA polymerase processivity factor in... -

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Basic information

Entry
Database: EMDB / ID: EMD-16925
TitleFocused refinement of HSV-1 DNA polymerase processivity factor in halted elongation state
Map data
Sample
  • Complex: HSV-1 DNA polymerase-processivity factor complex in halted elongation state
KeywordsDNA / Polymerase / Complex / TRANSFERASE
Biological speciesHuman alphaherpesvirus 1 strain KOS
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsGustavsson E / Grunewald K / Elias P / Hallberg M
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 28, 2023-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16925.png

Downloads & links

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Map

FileDownload / File: emd_16925.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.43398777 - 1.1699501
Average (Standard dev.)0.00002409145 (±0.0112133855)
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_16925_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_16925_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_16925_half_map_2.map
Projections & Slices
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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

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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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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
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: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / 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)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7luf


Chain - Source name: PDB / Chain - Initial model type: experimental model

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