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- PDB-7z0z: Abortive infection DNA polymerase AbiK from Lactococcus lactis, Y... -

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Basic information

Entry
Database: PDB / ID: 7z0z
TitleAbortive infection DNA polymerase AbiK from Lactococcus lactis, Y44F variant
ComponentsAbiK
KeywordsANTIVIRAL PROTEIN / DNA polymerase / protein-primed DNA synthesis / template-independent DNA synthesis / abortive infection
Function / homologyReverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily / metal ion binding / AbiK
Function and homology information
Biological speciesLactococcus lactis (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsFigiel, M. / Gapinska, M. / Czarnocki-Cieciura, M. / Zajko, W. / Nowotny, M.
Funding support Poland, European Union, 2items
OrganizationGrant numberCountry
Foundation for Polish SciencePOIR.04.04.00-00-20E7/16-00 Poland
European Regional Development FundPOIG.02.02.00-14-024/08-00European Union
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases.
Authors: Małgorzata Figiel / Marta Gapińska / Mariusz Czarnocki-Cieciura / Weronika Zajko / Małgorzata Sroka / Krzysztof Skowronek / Marcin Nowotny
Abstract: Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases. They are ...Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases. They are unique in the way they combine the ability to synthesize DNA in a template-independent manner with protein priming. Here, we report crystal and cryo-electron microscopy structures of two Abi polymerases: AbiK and Abi-P2. Both proteins adopt a bilobal structure with an RT-like domain that comprises palm and fingers subdomains and a unique helical domain. AbiK and Abi-P2 adopt a hexameric and trimeric configuration, respectively, which is unprecedented for reverse transcriptases. Biochemical experiments showed that the formation of these oligomers is required for the DNA polymerization activity. The structure of the AbiK-DNA covalent adduct visualized interactions between the 3' end of DNA and the active site and covalent attachment of the 5' end of DNA to a tyrosine residue used for protein priming. Our data reveal a structural basis of the mechanism of highly unusual template-independent protein-priming polymerases.
History
DepositionFeb 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 28, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 5, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AbiK
B: AbiK
C: AbiK
D: AbiK
E: AbiK
F: AbiK


Theoretical massNumber of molelcules
Total (without water)429,7036
Polymers429,7036
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22830 Å2
ΔGint-75 kcal/mol
Surface area131930 Å2
MethodPISA

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Components

#1: Protein
AbiK


Mass: 71617.109 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis (lactic acid bacteria)
Gene: abiK / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): Star / References: UniProt: Q48614

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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

ComponentName: Homohexamer of L. lactis AbiK Y44F mutant / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.42 MDa / Experimental value: NO
Source (natural)Organism: Lactococcus lactis (lactic acid bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Strain: Star
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1400 mMsodium chlorideNaCl1
220 mMHepesC8H18N2O4S1
31 mMdithiothreitolC4H10O2S21
40.5 mMethylenediaminetetraacetic acidC10H16N2O81
SpecimenConc.: 1.74 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18_3845: / Classification: refinement
EM software
IDNameVersionCategory
9PHENIX1.18-3845model refinement
13cryoSPARCv3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2306347
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50340 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00530132
ELECTRON MICROSCOPYf_angle_d0.58540614
ELECTRON MICROSCOPYf_dihedral_angle_d16.7073798
ELECTRON MICROSCOPYf_chiral_restr0.0474302
ELECTRON MICROSCOPYf_plane_restr0.0035184

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