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- PDB-8apm: Vaccinia virus DNA helicase D5 residues 323-785 hexamer with boun... -

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Basic information

Entry
Database: PDB / ID: 8apm
TitleVaccinia virus DNA helicase D5 residues 323-785 hexamer with bound DNA processed in C1
Components
  • DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')
  • DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')
  • Primase D5
KeywordsVIRAL PROTEIN / DNA helicase / D5_N domain / DUF5906 domain / Pox_D5 domain / SF3 helicase
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
DNA / DNA (> 10) / Uncoating factor OPG117
Similarity search - Component
Biological speciesVaccinia virus Copenhagen
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsBurmeister, W.P. / Hutin, S. / Ling, W.L. / Grimm, C. / Schoehn, G.
Funding support France, 5items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Structure summary / Category: chem_comp_atom / chem_comp_bond / struct / Item: _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Primase D5
E: Primase D5
D: Primase D5
B: Primase D5
C: Primase D5
F: Primase D5
X: DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')
Y: DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')


Theoretical massNumber of molelcules
Total (without water)339,4508
Polymers339,4508
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "F"
d_1ens_2chain "E"
d_2ens_2chain "C"
d_3ens_2chain "D"
d_4ens_2chain "B"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ALAGLUA1 - 359
d_21ens_1ALAGLUF1 - 359
d_11ens_2ALAILEB1 - 449
d_21ens_2ALAILEE1 - 449
d_31ens_2ALAILEC1 - 449
d_41ens_2ALAILED1 - 449

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(0.708786677805, 0.655702712323, 0.26014495655), (-0.3244525598, 0.630479313181, -0.705142802623), (-0.626380061787, 0.415391127378, 0.659619761295)-95.9514405775, 217.531052074, 86.7585181847
2given(0.151281351482, 0.972098850048, -0.179270126984), (-0.0610412304883, -0.171824296393, -0.983234651215), (-0.986604237207, 0.159687936001, 0.0333442981875)7.16756479212, 345.673399517, 281.534757899
3given(0.688973090306, 0.670209571407, 0.275926097403), (-0.325167025241, 0.626060384798, -0.708741702091), (-0.647751871113, 0.39858189244, 0.649268810655)-97.1312122557, 219.269140305, 94.4010023186
4given(-0.135476602955, 0.643199239248, -0.753618490141), (0.68922878501, -0.485247312355, -0.538050859832), (-0.711765250602, -0.592308859029, -0.377571772715)193.938658156, 206.143034882, 416.516106259

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Components

#1: Protein
Primase D5


Mass: 53495.285 Da / Num. of mol.: 6 / Mutation: L221A, D222M
Source method: isolated from a genetically manipulated source
Details: obtained after tobacco etch virus protease cleavage with 2 additional N-terminal residues from the cleavage site.
Source: (gene. exp.) Vaccinia virus Copenhagen / Strain: Copenhagen / Gene: D5R / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P21010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: DNA chain DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')


Mass: 9265.994 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: synthetic, at the current resolution the sequence does not matter, the DNA oligomer was end-labelled: biotin-ccgaatcaggaagataacagcggtttagcc3-digoxigenin
Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')


Mass: 9211.910 Da / Num. of mol.: 1 / Mutation: G2A / Source method: obtained synthetically
Details: At the current resolution, the base mismatch does not have any impact as it is located in an unpaired part of the DNA.
Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Primase D5 C-terminal fragment res. 323 - res. 785COMPLEXconstructall0MULTIPLE SOURCES
2Primase-helicase D5 fragment residues 323-785COMPLEXObtained after TEV (tobacco etch virus) cleavage of a construct#11RECOMBINANT
3DNA (5'-D(P*CP*CP*GP*AP*AP*TP*CP*A)-3')COMPLEX#21SYNTHETIC
4DNA (5'-D(P*TP*GP*AP*TP*TP*CP*GP*G)-3')COMPLEX#31SYNTHETIC
Molecular weightValue: 0.325 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Vaccinia virus Copenhagen10249
23Synthetic construct (others)32630
34Synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Spodoptera frugiperda (fall armyworm)7108SF21
23Synthetic construct (others)32630
34Synthetic construct (others)32630
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClC4H12NO3Cl1
250 mMsodium chlorideNaCl1
3100 mMpotassium chlorideKCl1
42 mMmagnesium chlorideMgCl21
50.5 %glycerolC3H8O31
610 mMbeta-mercaptoethanolHOCH2CH2SH1
SpecimenConc.: 0.24 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample contained also a dsDNA oligomer in slight stoechiometric excess over hexamers.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3000 nm
Image recordingElectron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 830
Image scansSampling size: 1.21 µm / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7Coot0.9.8.1model fitting
8UCSF Chimera1.15model fitting
10RELION3.1initial Euler assignment
11RELIONfinal Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 202659 / Details: Using 2D templates
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 26710 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 152 / Protocol: OTHER / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 304.4 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001621190
ELECTRON MICROSCOPYf_angle_d0.395128667
ELECTRON MICROSCOPYf_chiral_restr0.03663220
ELECTRON MICROSCOPYf_plane_restr0.00313576
ELECTRON MICROSCOPYf_dihedral_angle_d9.43298042
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints2.77481171111E-13
ens_2d_2BELECTRON MICROSCOPYNCS constraints2.13892782749E-10
ens_2d_3BELECTRON MICROSCOPYNCS constraints5.03524043816E-13
ens_2d_4BELECTRON MICROSCOPYNCS constraints4.60719289464E-13

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