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- EMDB-15575: Vaccinia virus DNA helicase D5 residues 323-785 hexamer with boun... -

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Basic information

Entry
Database: EMDB / ID: EMD-15575
TitleVaccinia virus DNA helicase D5 residues 323-785 hexamer with bound DNA processed in C1
Map dataMasked map in C1 from Relion PostProcess
Sample
  • Complex: Primase D5 C-terminal fragment res. 323 - res. 785
    • Complex: Primase-helicase D5 fragment residues 323-785
      • Protein or peptide: Primase D5
    • Complex: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')
      • DNA: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')
    • Complex: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')
      • DNA: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')
KeywordsDNA helicase / D5_N domain / DUF5906 domain / Pox_D5 domain / SF3 helicase / VIRAL PROTEIN
Function / homology
Function and homology information


helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / host cell cytoplasm / hydrolase activity / ATP binding
Similarity search - Function
DNA primase/nucleoside triphosphatase, C-terminal / Poxvirus D5 protein-like / Bacteriophage/plasmid primase, P4, C-terminal / D5 N terminal like / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Uncoating factor OPG117
Similarity search - Component
Biological speciesVaccinia virus Copenhagen / Synthetic construct (others) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsBurmeister WP / Hutin S / Ling WL / Grimm C / Schoehn G
Funding support France, 5 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-13-BSV8-0014 France
Agence Nationale de la Recherche (ANR)ANR-15-IDEX-02 France
Agence Nationale de la Recherche (ANR)ANR-10-INBS-0005-02 France
Agence Nationale de la Recherche (ANR)ANR-17-EURE-0003 France
Agence Nationale de la Recherche (ANR)PoxRep France
CitationJournal: Viruses / Year: 2022
Title: The Vaccinia Virus DNA Helicase Structure from Combined Single-Particle Cryo-Electron Microscopy and AlphaFold2 Prediction.
Authors: Stephanie Hutin / Wai Li Ling / Nicolas Tarbouriech / Guy Schoehn / Clemens Grimm / Utz Fischer / Wim P Burmeister /
Abstract: Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA ...Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323-785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.
History
DepositionAug 10, 2022-
Header (metadata) releaseNov 9, 2022-
Map releaseNov 9, 2022-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15575.png

Downloads & links

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Map

FileDownload / File: emd_15575.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked map in C1 from Relion PostProcess
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.21 Å/pix.
x 256 pix.
= 309.76 Å		1.21 Å/pix.
x 256 pix.
= 309.76 Å		1.21 Å/pix.
x 256 pix.
= 309.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.028547706 - 0.07065984
Average (Standard dev.)0.00023617754 (±0.0023804225)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 309.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map from Relion Refine3D in C1

Fileemd_15575_half_map_1.map
AnnotationHalf map from Relion Refine3D in C1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map from Relion Refine3D in C1

Fileemd_15575_half_map_2.map
AnnotationHalf map from Relion Refine3D in C1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Primase D5 C-terminal fragment res. 323 - res. 785

EntireName: Primase D5 C-terminal fragment res. 323 - res. 785
Components
  • Complex: Primase D5 C-terminal fragment res. 323 - res. 785
    • Complex: Primase-helicase D5 fragment residues 323-785
      • Protein or peptide: Primase D5
    • Complex: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')
      • DNA: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')
    • Complex: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')
      • DNA: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')

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Supramolecule #1: Primase D5 C-terminal fragment res. 323 - res. 785

SupramoleculeName: Primase D5 C-terminal fragment res. 323 - res. 785 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: construct
Molecular weightTheoretical: 325 KDa

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Supramolecule #2: Primase-helicase D5 fragment residues 323-785

SupramoleculeName: Primase-helicase D5 fragment residues 323-785 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Obtained after TEV (tobacco etch virus) cleavage of a construct
Source (natural)Organism: Vaccinia virus Copenhagen

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Supramolecule #3: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')

SupramoleculeName: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3') / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Synthetic construct (others) / Synthetically produced: Yes

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Supramolecule #4: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')

SupramoleculeName: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3') / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Synthetic construct (others) / Synthetically produced: Yes

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Macromolecule #1: Primase D5

MacromoleculeName: Primase D5 / type: protein_or_peptide / ID: 1
Details: obtained after tobacco etch virus protease cleavage with 2 additional N-terminal residues from the cleavage site.
Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Vaccinia virus Copenhagen / Strain: Copenhagen
Molecular weightTheoretical: 53.495285 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: AMGNKLFNIA QRILDTNSVL LTERGDYIVW INNSWKFNSE EPLITKLILS IRHQLPKEYS SELLCPRKRK TVEANIRDML VDSVETDTY PDKLPFKNGV LDLVDGMFYS GDDAKKYTCT VSTGFKFDDT KFVEDSPEME ELMNIINDIQ PLTDENKKNR E LYEKTLSS ...String:
AMGNKLFNIA QRILDTNSVL LTERGDYIVW INNSWKFNSE EPLITKLILS IRHQLPKEYS SELLCPRKRK TVEANIRDML VDSVETDTY PDKLPFKNGV LDLVDGMFYS GDDAKKYTCT VSTGFKFDDT KFVEDSPEME ELMNIINDIQ PLTDENKKNR E LYEKTLSS CLCGATKGCL TFFFGETATG KSTTKRLLKS AIGDLFVETG QTILTDVLDK GPNPFIANMH LKRSVFCSEL PD FACSGSK KIRSDNIKKL TEPCVIGRPC FSNKINNRNH ATIIIDTNYK PVFDRIDNAL MRRIAVVRFR THFSQPSGRE AAE NNDAYD KVKLLDEGLD GKIQNNRYRF AFLYLLVKWY KKYHVPIMKL YPTPEEIPDF AFYLKIGTLL VSSSVKHIPL MTDL SKKGY ILYDNVVTLP LTTFQQKISK YFNSRLFGHD IESFINRHKK FANVSDEYLQ YIFIEDISSP

UniProtKB: Uncoating factor OPG117

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Macromolecule #2: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')

MacromoleculeName: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3') / type: dna / ID: 2
Details: synthetic, at the current resolution the sequence does not matter, the DNA oligomer was end-labelled: biotin-ccgaatcaggaagataacagcggtttagcc3-digoxigenin
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.265994 KDa
SequenceString:
(DC)(DC)(DG)(DA)(DA)(DT)(DC)(DA)(DG)(DG) (DA)(DA)(DG)(DA)(DT)(DA)(DA)(DC)(DA)(DG) (DC)(DG)(DG)(DT)(DT)(DT)(DA)(DG)(DC) (DC)

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Macromolecule #3: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')

MacromoleculeName: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3') / type: dna / ID: 3
Details: At the current resolution, the base mismatch does not have any impact as it is located in an unpaired part of the DNA.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.21191 KDa
SequenceString:
(DG)(DG)(DC)(DT)(DT)(DA)(DG)(DT)(DC)(DC) (DT)(DT)(DC)(DT)(DA)(DT)(DT)(DG)(DT)(DC) (DG)(DC)(DA)(DG)(DA)(DT)(DT)(DC)(DG) (DG)

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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

Concentration0.24 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
50.0 mMC4H12NO3ClTris-HCl
50.0 mMNaClsodium chloride
100.0 mMKClpotassium chloride
2.0 mMMgCl2magnesium chloride
0.5 %C3H8O3glycerol
10.0 mMHOCH2CH2SHbeta-mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
DetailsThe sample contained also a dsDNA oligomer in slight stoechiometric excess over hexamers.

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 830 / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 1.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 202659 / Details: Using 2D templates
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 26710
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 3 / Avg.num./class: 25000 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 152
Output model

PDB-8apm:
Vaccinia virus DNA helicase D5 residues 323-785 hexamer with bound DNA processed in C1

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