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- PDB-7r06: Abortive infection DNA polymerase AbiK from Lactococcus lactis -

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Basic information

Entry
Database: PDB / ID: 7r06
TitleAbortive infection DNA polymerase AbiK from Lactococcus lactis
Components
  • AbiK
  • DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
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 / DNA / DNA (> 10) / AbiK
Function and homology information
Biological speciesLactococcus lactis (lactic acid bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.27 Å
AuthorsFigiel, M. / Nowotny, M. / Gapinska, M. / Czarnocki-Cieciura, M. / Zajko, W.
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 1, 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
G: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
B: AbiK
H: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
C: AbiK
I: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
D: AbiK
J: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
E: AbiK
K: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
F: AbiK
L: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)450,83012
Polymers450,83012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
AbiK


Mass: 71713.086 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 (E. coli) / References: UniProt: Q48614
#2: DNA chain
DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')


Mass: 3425.224 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
Has ligand of interestN

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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
IDNameTypeEntity IDParent-IDSource
1Homohexamer of AbiK with single-stranded DNA covalently attached to each of the subunitsCOMPLEXall0MULTIPLE SOURCES
2Abortive infection protein AbiKCOMPLEX#11RECOMBINANT
3DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')COMPLEX#21NATURAL
Molecular weightValue: 0.42 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Lactococcus lactis (lactic acid bacteria)1358
33Escherichia coli (E. coli)562
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1400 mMsodium chlorideNaCl1
220 mMHepesC8H18N2O4S1
31 mMdithiothreitolC4H10O2S21
40.5 mMethylenediaminetetraacetic acidC10H16N2O81
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18_3845: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 227461 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00632160
ELECTRON MICROSCOPYf_angle_d0.78643566
ELECTRON MICROSCOPYf_dihedral_angle_d21.9134518
ELECTRON MICROSCOPYf_chiral_restr0.0534656
ELECTRON MICROSCOPYf_plane_restr0.0055364

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